2017 CSU Biotechnology Symposium
Posters with Author Listings and Abstracts
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Poster #: 1
Campus: CSU Northridge
Poster Category: Agriculture/Biofuels/Environment
Keywords: Arabidopsis thaliana, Genetic engineering, Weather pollution
Project Title: Overexpression of an aminocyclopropane-1-carboxylic acid synthase gene in Arabidopsis thaliana and its effect on removing environmental pollutants
Author List:
Ramadoss, Niveditha; Graduate, Biology, California State University, Northridge, Presenting Author
Gupta, Dinesh; Washington University, St. Louis
Guenther, Alex; University of California, Irvine
Basu, Chhandak; Biology, California State University, Northridge
Abstract: Rise in air pollution has led to increasing cancer, several respiratory and cardiac illnesses in humans. World Health Organization estimates that one in eight person die from air pollution. The main objective of our study is to analyze the feasibility of genetically engineered plants to remove polluting volatile compounds from air. To achieve this goal we genetically transformed Arabidopsis thaliana with an ACS (Aminocyclopropane-1-carboxylic acid synthase) gene. Using quantitative real-time PCR (qPCR), we previously confirmed that the ACS gene was upregulated in poplar (Populus trichocarpa) plants when exposed to environmental pollutants including ozone and methyl vinyl ketone. Therefore, we hypothesize that ACS gene might play an important role in poplar plants in mitigating stresses resulting from pollutant exposure. The homolog of poplar ACC synthase gene was PCR amplified from Arabidopsis thaliana and cloned into pBINmgfp5-er vector, featuring a constitutive cauliflower mosaic virus promoter removing the native jellyfish green fluorescent (GFP) gene. The recombinant plasmid containing the ACS gene was electroporated into the GV101 strain of Agrobacterium tumefaciens. Arabidopsis plants were genetically engineered with ACS gene by dipping the T0 (transgenic zero or wild type) flowering plants into GV101 strain of Agrobacterium containing the recombinant binary vector. The transformed plants were selected through kanamycin resistance and grown till T3 (third transgenic) generation. The ACS gene expression of these plants will be confirmed through qPCR. The pollutant absorbing capacity will be tested by placing the transformed T3 plants in an environmental gas chamber where they will be exposed to various levels of pollutants. The ability of the transformed plants to absorb pollutants will be evaluated by performing gas chromatography mass spectrometry analyses to measure pollutants in the inlet and outlet airflow in order to quantify the pollutant deposition rate. Our use of a model plant Arabidopsis is ‘proof of concept’. Our future goal will be to transform tree species and evaluate their potentials to remove pollutants. These studies will open doors for use of transgenic plants to clean up environments.
Poster #: 2
Campus: Sonoma State University
Poster Category: Agriculture/Biofuels/Environment
Keywords: Biosensors, Wireless Biosensor Network , Soil
Project Title: A Comparative Study of RF Wave Attenuation in Soil and Sand
Author List:
Palmerin, Abraham; Undergraduate, Engineering Science , Sonoma State University, Presenting Author
Farahmand, Farid; Engineering Science, Sonoma State University
Fong, Erin; Lawrence Berkeley National Laboratory, Engineering Division
Abstract: Soil is the most spatially complex stratum of a terrestrial ecosystem. Therefore, soil monitoring and understanding changes in soil ecosystem are considered to be important aspects of any environmental monitoring. The recent emergence of Wireless Biosensor Networks (WBNs) promises addressing many ecological questions, particularly related to soil, though providing uninterrupted real-time data from various biosensors using RF signals.
In this work we focused on understanding the impact of soil composition (e.g., clay content) as RF waves travel underground. This study is crucial in order to define the appropriate RF band, power level, and RF antenna size for extended underground monitoring.
Our research focused on 5.0, 2.5, and 0.9 GHz ISM bands. The first part of our experiment was conducted in a 103-gallon weathertight trunk container filled with sand, mostly composed of silica in the form of quartz, with no clay. We measured the RF signal path loss through the sand for the above frequencies at different horizontal distances between the transmitter and receiver. We also measured RF signal strength as we changed the vertical distances between the two antennas. In the second part of our experiment, we used regular gardening soil mostly composed of organic matter, perlite and vermiculite. Vermiculite keeps the soil hydrated and is part of the clay minerals group. We made similar measurements. In both cases, we maintained the temperature at 23 degrees Celsius. We normalized the obtained path loss measurements as a function of distance for all frequencies to remove any offset difference due to factors such as antennas.
Our results indicate that at 0.9 GHz the received signal power level decays with distance by about 1.6 dB/cm when traveling in soil. We also found that at higher frequencies, the RF signal experiences 4-6 dB more attenuation in soil than air. The exact value depends on the frequency and antenna polarization type. On the other hand, the path loss in sand is about 0.9-1.2 dB more than air. It is therefore, preferred to use lower band frequencies for underground biosensors. However, the trade off will be antenna size and its design complexity. Our results conclude that it is critical to carefully manage signal power and understand antenna design when wireless biosensors are placed in an area with high quantity of clay content. This study was carried out in collaboration with Lawrence Berkeley National Laboratory, Division of Earth Sciences.
Poster #: 3
Campus: CSU Long Beach
Poster Category: Agriculture/Biofuels/Environment
Keywords: Arabidopsis, Senescence, Epigenetics
Project Title: A TIR-NBS-LRR Class Disease Resistance Protein Mays be a Positive Regulator of Developmental Senescence in Arabidopsis thaliana
Author List:
Mendoza, Victor ; Undergraduate, Chemistry & Biochemistry, California State University, Long Beach, Presenting Author
Brusslan, Judy; Biological Sciences, California State University, Long Beach
Abstract: Leaf senescence is the final stage in leaf development in which the leaf reallocates its nutrients to growing parts of the plant. This study aims to determine if the At4g14370 gene, which encodes a TIR-NBS-LRR class disease resistance protein (DRP), is involved in leaf senescence in Arabidopsis thaliana. This gene is up-regulated in leaf senescence and the increase in expression occurs in parallel to increases in the H3K4me3 mark. Two distinct transfer-DNA (T-DNA) insertion lines that disrupt At4g14370, k4-drp-1 and k4-drp-2, have been isolated and their location has been determined by DNA sequencing. Loss of gene expression was confirmed by amplification of cDNA using insertion-specific primers. Chlorophyll and total protein levels were used as physical markers for age. Differences between wild type and mutant leaves were quantified and compared using two-tailed student’s t-test. The transcript levels of robust senescence-up regulated genes (WRKY75 and NIT2), were also compared. Our results revealed a significant increase of protein levels in the k4-drp-1 mutant when compared to wild type. A trend of increased chlorophyll levels in k4-drp mutants were also observed when compared to wild type. Quantitative real-time PCR data revealed no trends of increased senescence associated gene transcript levels. We infer that the TIR-NBS-LRR, from the TNL-H subfamily, K4-DRP, may be involved in the protein degradation signaling cascade in senescence. Further work includes generating triple mutants of the TNL-H class genes that are up-regulated during senescence and marked by H3K4me3. A strong delayed-senescence phenotype in triple TNL-H mutants would suggest that the TNL-H subfamily of genes may be positive regulators of senescence.
Poster #: 4
Campus: CSU Fresno
Poster Category: Agriculture/Biofuels/Environment
Keywords: fleabane, herbicide resistance, greenhouse common garden
Project Title: Fitness cost of glyphosate resistance in hairy fleabane (Erigeron bonariensis) under drought conditions
Author List:
Pathak, Ankit; Graduate, Biology, California State University, Fresno, Presenting Author
Waselkov, Katherine; Biology, California State University, Fresno
Abstract: Many agriculturally invasive, weedy plant species have evolved resistance various chemical herbicides, in large part because of continuous application of these chemicals to agricultural environments. Glyphosate is a powerful, low-toxicity herbicide that has been extensively applied to crops in the San Joaquin Valley of California since the 1990s. In 2007, a glyphosate-resistant population of hairy fleabane (Erigeron bonariensis) was discovered on a California roadside, and since then, glyphosate resistance in fleabane has become widespread in the San Joaquin Valley. During the same time period (2008 to the present), extreme drought conditions became a major factor in California agriculture. Since this weed species competes with young fruit trees and vines for resources, and as water resources become more limited due to climatic changes, our lab has begun to study the interaction between glyphosate resistance (GR) and drought stress in hairy fleabane. Our hypothesis was that glyphosate resistance has no fitness cost under drought-stressed conditions. Twenty plants each from a well-characterized GR fleabane population and a glyphosate-sensitive (GS) population were grown in a greenhouse environment: half of the plants of each biotype were exposed to drought stress conditions (40% field capacity) from transplantation at the 4-leaf stage until seed production. We measured different fitness components, including height and leaf number over time, days to bolting and flowering, above-ground and below-ground biomass, and seed production. Treatment had a stronger effect than biotype on all measurements that showed significant differences between groups, including days to flowering (8.75 days earlier in drought-stressed plants), and height and leaf production in younger plants (greater in the control group in growth weeks 1-3). However, there was a non-significant trend towards a reversal of the relative fitness of GR and GS plants in drought conditions: GR plants, which were consistently taller in control conditions, were shorter than GS plants under water stress at all time points. Our results suggest that, contrary to our hypothesis, glyphosate resistance has a small fitness cost in fleabane under drought-stress conditions, which might be detectable with a larger sample size in repeated experimentation, soon to be conducted in our lab. Our future experiments may have implications for integrated pest management (IPM) in Central Valley agriculture.
Poster #: 5
Campus: Humboldt State University
Poster Category: Agriculture/Biofuels/Environment
Keywords: Curriculum Development, antibiotics, biodiversity
Project Title: More biodiversity is seen in Streptomyces isolates from diverse ecosystem types than in isolates from similar but geographically distant sites.
Author List:
Stoflet, Terilyn; Undergraduate, Biology, Humboldt State University, Presenting Author
Wilson, Mark; Biology, Humboldt State University
Abstract: Understanding factors that govern microbial diversity and distribution will help optimize strategies to isolate microorganisms that produce novel bioactive compounds. A recent study compared sequences of 5 protein-coding genes (atpD, trpB, gyrB, rpoB and recA) in 191 bacteria from the genus Streptomyces that were isolated from grassland soils across the US and Canada. Identical alleles of the genes were often present in isolates collected thousands of kilometers apart at similar latitudes. Some allelic variation was observed in isolates from distinct longitudes. We tested the hypothesis that alternative ecosystems might harbor biodiversity not sampled in grassland soils. As part of a course-based research project, we isolated 50 antibiotic-producing bacteria from air and soil samples in a coastal redwood forest. Sequencing of PCR-amplified 16S rRNA genes revealed that 25 of our isolates were from the genus Streptomyces, and 20 of these fell into the 6 phylotypes examined in the previous study. We extracted genomic DNA, PCR-amplified and sequenced atpD, trpB, gyrB, rpoB and recA, and analyzed sequence alignments. Alleles from our isolates were rarely identical to those found in the grassland isolates, and pairwise comparisons with grassland isolates often exhibited higher levels of non-identity than seen in any pairwise comparison of grassland alleles. Although the previous study found that rapid, ongoing dispersal of Streptomyces erased traces of endemism, our results suggest that unique selective factors in alternative ecosystems might overcome dispersal effects. Our results suggest that novel bioactive compounds might be more readily found by searching unique ecosystem types rather than geographically separated ecosystems. We have developed a syllabus and protocols to further this research as a course-based research project carried out by students enrolled in an introductory Biology course, and 500 students will isolate and characterize antibiotic-producing microorganisms as part of this project at HSU in 2017. Student attitudes and persistence to graduation will be monitored to determine if participating in an authentic course-based research project improves student outcomes. We are actively searching for partner schools that would be interested in implementing the project on their campuses. This work was supported by curricular materials developed as part of the Small World Initiative at Yale University and a Curriculum Development grant from CSUPERB.
Poster #: 6
Campus: Sonoma State University
Poster Category: Agriculture/Biofuels/Environment
Keywords: wastewater treatment, anaerobic digestion, microbial fuel cells
Project Title: A microbial fuel cell-based system for treating winery wastewater
Author List:
Sacher, Gabriel; Undergraduate, Biology, Sonoma State University, Presenting Author
Kozlowski, John; Graduate, Biology, Sonoma State University
Simpson, David; Okinawa Institute of Science and Technology, Biological Systems Unit
Goryanin, Igor; Okinawa Institute of Science and Technology, Biological Systems Unit
Farahmand, Farid; Engineering Science, Sonoma State University
Cohen, Michael; Biology, Sonoma State University
Abstract: Wastewater treatment can be a major cost for wineries. The most energy-intensive portion of the treatment process is aerating the wastewater to facilitate microbial oxidation of organic compounds. However, some bacteria, instead of consuming oxygen, are able to oxidize organics by delivering electrons to conductive surfaces. Microbial fuel cells (MFCs) direct this flow of electrons to an external cathode, generating an electric current, and to methanogens that reside within the anodic chamber, generating biogas. We are investigating applications of MFC technology as an energy-saving substitute for wastewater aeration. The entire system, located at Vintners Square, Santa Rosa, CA, consists of a mixing tank, two novel tubular MFCs operated in parallel, and a sand filter, all maintained at ambient temperature. Outflow from the system is used to drip-irrigate on-site landscaping. Concentrations of organics in the wastewater were measured as chemical oxygen demand (COD). Continuous feeding of 0.2 to 2.5 kg COD/m^3 d to the MFCs gave removal rates ranging from 0.1 to 2.3 kg COD/m^3 d. Energy in the consumed COD was converted primarily to biogas, with external electrical current serving as an indicator of MFC health. Results gathered from the testing and optimization of this pilot system will inform the development of full-scale MFC-based systems to treat winery wastewater to a level suitable for irrigation.
Poster #: 7
Campus: CSU Stanislaus
Poster Category: Agriculture/Biofuels/Environment
Keywords: PHB degradation, bioplastics, induction, ,
Project Title: Characterization of Bacteria Capable of Degrading Polyhydroxybutyrate
Author List:
Vera, Hector; Undergraduate, Biological Sciences, California State University, Stanislaus, Presenting Author
Khut, Melanie; Undergraduate, Biological Sciences, California State University, Stanislaus, Presenting Author
Thao, My Lo; Biological Sciences, California State University, Stanislaus
Abstract: Petroleum-based plastics are an important part of our lives. Unfortunately, they are made from non-renewable resources and are recalcitrant to degradation in the environment or through artificial means. As a result, current research has been aimed at finding alternative sources, and one such source is polyhydroxybutyrate (PHB). PHB is a biodegradable and biocompatible polymer that is a principle component of bioplastics. However, in order to ensure that the bioplastics made from PHB degrade efficiently, more research on the bacteria that break down PHB is needed. Previously, we have isolated and identified fourteen unique bacterial species capable of degrading PHB from different sources, the majority of which were Pseudomonas species. The objectives of the current research are to characterize the PHB-degrading bacteria by determining their optimum growth temperature, nutrient requirements, and induction of the genes involved in PHB degradation. The optimum growth temperature was determined by growing the bacteria in liquid media at various temperatures and measuring growth using turbidimetry. To determine whether the isolates can use PHB as a source of carbon, prototrophy tests were done. Induction studies were carried out on Pseudomonas alcaliphila. Chemicals tested were PHB, 3-hydroxybutyric acid (3HB), glucose, and lactic acid and samples were taken at 4, 8, 12, and 16 hours following induction. Combinations of PHB+glucose and 3HB+glucose were also used to test for catabolite repression. The optimum temperatures for the majority of the isolates were between 32° C and 36° C with generation times ranging from 48 to 203 minutes. Pseudomonas alcaliphila had one of the shortest generation time of 48 minutes at 36° C. Five of the isolates could use PHB as a sole carbon source. The best inducer of the PHB gene was PHB and it was fully induced at 8, 12, and 16 hour induction. 3HB was a less effective inducer than PHB and there was no induction with lactate or glucose. Catabolite repression was observed with 3HB+glucose but not with PHB+glucose. The next step will be to clone and sequence the gene(s) and characterize the enzyme(s) involved in PHB degradation.
Poster #: 8
Campus: Cal Poly Pomona
Poster Category: Astrobiology
Keywords: astrobiology, permafrost, metabolome
Project Title: Biochemical Characterizations of Arctic Permafrost Along an Age Gradient
Author List:
Giatpaiboon, Scott; Graduate, Chemistry & Biochemistry, California State Polytechnic University, Pomona, Presenting Author
Mackelprang, Rachel; Biology, California State University, Northridge
Mogul, Rakesh; Chemistry & Biochemistry, California State Polytechnic University, Pomona
Abstract: Studies on the microbiology of Arctic permafrost clearly show that microbial life thrives at anaerobic subzero temperatures. As such, permafrost environments serve as excellent terrestrial analogs for the Mars subsurface, which contains a globally distributed permafrost and experiences prolonged subzero temperatures. Phylogenetic studies on permafrost obtained from Alaskan loess soils suggest that both microbial diversity and abundance decrease along a geochronosequence gradient of 5 to 35 kyr, with the Firmicutes being among the most abundant phyla at the older ages. In this poster presentation, we will present the current status of our complementary work focused on biochemical characterizations along the geochronosequence gradient. As proxies for oxidative stress and microbial abundance, we measured the bulk catalase specific activities and ATP abundances, respectively, in samples collected at 12, 25, and 35 kyr along the gradient. Catalase specific activities, as measured by volume displacement and calculated per gram of permafrost (n=6), were quite similar across the transect with relative values of 0.82 ± 0.03, 0.69 ± 0.11, and 0.87 ± 0.02 µkat/g, respectively. These results suggest that the permafrost microbiomes, regardless of sample age, experience similar levels of oxidative stress. In stark contrast, ATP abundances, as measured by luminescence (n=2), decreased as age of the permafrost increased, where both the total and intracellular ATP were highest in the 12 kyr samples and significantly lower in the 25 and 35 kyr samples (12 kyr > 25 kyr = 35 kyr). In context, these results are consistent with decreases in microbial abundance, including higher relative abundances of sporulated Firmicutes. Crucially, when expressing the catalase specific activity as enzyme activity per ATP (µkat/ATP RLU), the highest values were obtained from the oldest permafrost samples to yield a trend of 35 kyr = 25 kyr > 12 kyr. Though preliminary, these ratios do suggest that higher catalase expression levels and/or kinetically more active catalases are found in the older permafrost samples, regardless of vegetative or sporulated cell content. In conclusion, the biological contexts of these results will be discussed further, as will our plans to measure changes in the community metabolome across the gradient using gas chromatography-mass spectrometry.
Poster #: 9
Campus: CSU San Bernardino
Poster Category: Astrobiology
Keywords: Archaea, Thermophile, Hybridization
Project Title: Optimization of Group 1 Aigarchaeota-Specific Oligonucleotide Probes Using Clone-FISH
Author List:
Mosier, Damon; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Alvarado, Toshio; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Dodsworth, Jeremy; Biology, California State University, San Bernardino
Abstract: Aigarchaeota, a deeply branching lineage in the domain Archaea with no cultivated representatives, includes both thermophilic and hyperthermophilic microorganisms that reside in terrestrial and marine geothermal environments. This archaeal phylum contains nine proposed genus-level groups that have been confirmed via 16S rRNA sequencing, with Group 1 Aigarchaeota (Aig G1) being the focus of this study. To better detect and quantify Aig G1 in natural samples and enrichment cultures, Clone-FISH (fluorescence in situ hybridization) techniques were used to test possible oligonucleotide probes and to optimize hybridization conditions for FISH. A near-full length 16S rRNA gene obtained from Aig G1 was cloned into the plasmid pGEM-T. This construct was transformed into E.coli JM109(DE3) and the Aig G1 16S rRNA was subsequently induced by treatment with isopropyl beta-D-1-thiogalactopyranoside (IPTG) and chloramphenicol. FISH was then conducted with probes targeting the Aig G1 16S rRNA. Fluorescence signal intensities were observed using an epifluorescence microscope for induced and uninduced samples, as well as for a number of negative controls, at various formamide concentrations with fluorescently-labeled bacterial-specific (positive control for hybridization), archaeal-specific, and ‘Aigarchaeaota’-specific oligonucleotide probes. Induced, but not uninduced, cells were positive for the Archaea-specific probe, indicating successful induction of the Aig G1 16S rRNA gene transcript. A previously published Aig G1-specific probe showed specificity but had a relatively low signal intensity in comparison to the archaeal probe at all formamide concentrations, suggesting poor binding of this probe. Two other newly developed ‘Aigarchaeaota’ (G1)-specific probes were observed and compared at five different formamide concentrations, one of which (Aig G1 180 Cy3) was found to have low intensity and the other (Aig G1 1012 Cy3) that was at least 4-fold brighter than the previously published probe, with apparent optimal hybridization conditions at 30% formamide. This work will allow for better specific, sensitive detection of Aig G1 in natural samples and enrichment cultures, and will facilitate use of FISH coupled with nano-scale stable isotope mass spectrometry (nano-SIMS) to track the uptake of 13C labeled compounds by Aig G1 in future studies of their catabolic capabilities.
This work was supported by NSF grant DEB-1557042.
Poster #: 10
Campus: CSU Long Beach
Poster Category: Astrobiology
Keywords: astrobiology, microbial ecology, metal cycline
Project Title: Manganese-oxidizing bacteria from modern stromatolites in Pisa Pond, Death Valley National Park
Author List:
Levish, Michelle; Graduate, Biological Sciences, California State University, Long Beach, Presenting Author
Marquez, Tanya; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
McLain, Nathan; Graduate, Biological Sciences, California State University, Long Beach
Dillon, Jesse; Biological Sciences, California State University, Long Beach
Abstract: Pisa Pond is a small, saline pool on the eastern edge of the Badwater Basin in Death Valley National Park. It hosts unusual microbialites with layers of calcite, gypsum and manganese oxides that precipitate during alternating wet and dry seasons. The manganese oxides are thought to result from microbial metabolism of the biofilm that coats the submerged sections of these structures. These microbialites are modern analogs of early Earth communities and as such are of astrobiological interest. The aim of this study was to determine the microbial community present at this site by genomic and metagenomic sequencing, identify and characterize bacteria potentially responsible for the formation of the manganese oxide layer via targeted cultivation methods, and to determine whether the isolates are associated with the manganese-oxide layer of the stromatolite via FISH. 16S rRNA gene sequencing revealed a diverse community that varies with depth, with Alphaproteobacteria, Acidobacteria and Verrucomicrobia dominating the shallowest section, and Deltaproteobacteria and Deferribacteres with increased abundance in the deeper layers. One Mn(II)-oxidizing isolate has been obtained and was identified as a strain of Photobacterium. This isolate oxidizes Mn(II) only when in biofilms. The genome of this isolate has been sequenced using the MinION and will be used to screen for potential Mn(II) oxidation genes. This study will provide important insights into this novel microbialite system.
Poster #: 11
Campus: Sonoma State University
Poster Category: Astrobiology
Keywords: lignocellulose degradation, biofuel, astrobiology
Project Title: Bioprospecting alkaline springs for lignocellulose-degrading enzymes: A xylanase from Cellulomonas sp. strain FA1
Author List:
Gray, Justine; Graduate, Biology, Sonoma State University, Presenting Author
Kainuma, Mami; Biology, Sonoma State University
Hu, Ping; Lawrence Berkeley National Laboratory
Nguyen, My Vu; Lawrence Berkeley National Laboratory
Kamennaya, Nina; Lawrence Berkeley National Laboratory
Holman, Hoi-Ying; Lawrence Berkeley National Laboratory
Torok, Tamas; Lawrence Berkeley National Laboratory
Cohen, Michael; Biology, Sonoma State University
Abstract: The polysaccharides cellulose and hemicellulose in the cell-walls of terrestrial plants are the most abundant carbon source on Earth, composed of energy-rich sugars that can be used for the production of bioethanol by microbial biocatalysts. Currently, hemicellulose is discarded as a waste byproduct by paper mills and in the production of cellulosic bioethanol. However, with advances in processing, hemicellulose could be repurposed as a cheap and abundant feedstock for bioethanol production. Production of plant based biofuels is hindered by the presence of lignin, which must be degraded by pretreatment, such as by exposure to high pH, before hydrolytic enzymes can gain access to cell-wall polysaccharides. We are seeking to develop ways to utilize hemicellulose as a biofuel feedstock by applying alkaline-tolerant organisms and enzymes capable of degrading plant biomass and thereby consolidate bioprocessing steps for producing bioethanol. One potential source of such organisms are rare alkaline springs, similar to the alkaline vents where life may have originated, found at The Cedars, Sonoma, CA, in which plant matter falling into the Ca(OH)2-rich pH 11.5-11.9 springs is exposed to conditions that mimic those of industrial lignocellulose pretreatment. From these springs we isolated Cellulomonas sp. strain FA1, an ethanol-producing alkaline tolerant bacterium capable of degrading cellulose and hemicellulose and fermenting their component sugars. Sequence analysis of the strain FA1 genome revealed 82 glycosyl hydrolases. β-1,4-endoxylanase cleaves fermentable xylose residues from xylan, one of the primary components of hemicellulose. A gene from strain FA1 encoding a putative 36.2 kDa β-1,4-endoxylanase (FA1-XYL) was cloned into an IPTG-inducible vector and expressed in E. coli DH5α and the protein purified by Ni-NTA affinity chromatography. Enzyme kinetics and pH and temperature profiles will be determined by using the purified FA1-XYL enzyme in activity assays with xylan derived from beechwood as a substrate. The results from these experiments may elucidate potential applications of strain FA1 for advancing biofuel technology.
Poster #: 12
Campus: Cal Poly Pomona
Poster Category: Astrobiology
Keywords: astrobiology, planetary protection, extremophiles
Project Title: Metabolomics and oxidative extremtolerance of spacecraft-associated Acinetobacter grown on ethanol
Author List:
Baki, Ryan; Graduate, Chemistry & Biochemistry, California State Polytechnic University, Pomona, Presenting Author
Lee, Sooji; Graduate, Chemistry & Biochemistry, California State Polytechnic University, Pomona
Campos, Alexa ; Undergraduate, Chemistry & Biochemistry, California State Polytechnic University, Pomona
Perkins, Nicole ; Undergraduate, Chemistry & Biochemistry, California State Polytechnic University, Pomona
Barding, Gregory; Chemistry & Biochemistry, California State Polytechnic University, Pomona
Mogul, Rakesh ; Chemistry & Biochemistry, California State Polytechnic University, Pomona, Presenting Author
Abstract: Protecting Mars from biological contamination is critical to ensuring the integrity of future life-detection missions. Despite the stringent cleaning protocols for Mars-bound spacecraft, there remains a persistent bioburden within the cleanroom facilities where spacecraft are assembled. Typically, these spacecraft-associated isolates are tolerant towards oxidative, radiation, and desiccating conditions, and hence carry the potential to survive or persist on/in spacecraft during Mars exploration, and perhaps in the martian regolith. Among the most common genera found in spacecraft assembly facilities are the Acinetobacter; where molecular genetic studies suggest proliferation of this genus during spacecraft assembly. Herein, we describe molecular and biological studies that support the hypothesis that spacecraft cleaning reagents serve as carbon sources for the spacecraft-associated Acinetobacter. Cultivation and plate count assays clearly show that strains of Acinetobacter isolated from the Mars Odyssey and Mars Phoenix facilities grew on ethanol, a spacecraft cleaning reagent, when present as a sole carbon source under minimal conditions (0.2x M9, 26 µM Fe(II)). Under these conditions, a maximum growth rate of 0.45/h at ~10 mM ethanol was obtained for A. radioresistens 50v1, which was isolated for the surface of the Mars Odyssey. Metabolomic studies on this strain, using gas chromatography-mass spectrometry (GC-MS), show that cultures grown on 13C-ethanol yielded fully 13C-labeled metabolites including TCA/glyoxylate cycle intermediates, amino acids, and carbohydrates. Further, microbiology experiments demonstrate an oxidative extremotolerance under minimal conditions, with exposures of 10 mM hydrogen peroxide yielding ~2-log reductions from ~10^8 cfu/mL. Interestingly, these survivals increased ~10-fold when Mn(II) was present as the sole transition metal. In terms of survival mechanisms, our enzymology studies suggest that ethanol metabolism and oxidative extremotolerance involve a membrane-bound NAD/PQQ-dependent alcohol dehydrogenase and membrane-bound Fe-heme catalase, respectively. In conclusion, our student-centered work provides a biochemical rationale to the observed microbial ecology dynamics of spacecraft assembly facilities and, due to the observed oxidative extremotolerance under oligotrophic conditions, support the characterization of the spacecraft-associated Acinetobacter as potential forward contaminants of Mars.
Poster #: 13
Campus: CSU Fullerton
Poster Category: Astrobiology
Keywords: manganese, bacterial isolation, astrobiology
Project Title: Isolation and Identification of Novel Manganese Oxidizing Bacteria
Author List:
Phelan, Joshua; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Johnson, Hope; Biological Science, California State University, Fullerton
Abstract: Microbial manganese oxidation plays an important role in the biogeochemical cycling of manganese. Microbe mediated conversion of soluble Mn(II) to insoluble Mn(IV) results in greater deposition of Mn oxides than can be explained by abiotic factors alone. Biotic rates of Mn oxidation may be used in concordance with geological data to better piece together the history of life. Biogenic manganese oxides have also been shown to lower heavy metal concentrations in the water column by sequestering metal ions within manganese oxides. Manganese oxidizing microbes are phylogenetically diverse and have been isolated from many environments including fresh and saltwater environments, caves, and desert varnish. In this study, we attempt to isolate new manganese oxidizing bacteria from environmental samples taken from Little Hot Creek, a hot spring in the Long Valley Caldera near Mammoth Lake California. Environmental samples were plated on media variants designed for enrichment of manganese oxidizing bacteria. Each media variant was amended with 100 µM Mn(II) which allows for easy identification of manganese oxidizing colonies as solid manganese oxides are visibly brown. After isolation, suspected manganese oxidizing colonies were spot tested with LBB (Leucoberbelin Blue) dye for verification of manganese oxidation (LBB turns from light blue to dark blue when oxidized by manganese oxides). Colony PCR was performed on isolates to amplify the 16S rRNA gene, a phylogenetic marker, for sequencing. Sequences were aligned against those recorded in the NCBI database using the Basic Local Alignment Search Tool (BLAST). Thus far, four species of manganese oxidizing bacteria have been identified. These include two Sphingomonas spp., one Pseudomonas spp., and an Azoarcus spp. a β-proteobacteria that has not yet been demonstrated to oxidize manganese. This study was supported by the 2016 CSUPERB Presidents’ Commission Scholars Program.
Poster #: 14
Campus: Cal Poly Pomona
Poster Category: Astrobiology
Keywords: spectroscopy, synchrotron, astrobiology
Project Title: Spectroscopic Techniques for Studying Complex Enzyme Systems
Author List:
Phu, Phan N.; Undergraduate, Chemistry & Biochemistry, California State Polytechnic University, Pomona
Stieber, S. Chantal E.; Chemistry & Biochemistry, California State Polytechnic University, Pomona, Presenting Author
Abstract: Enzymes are able to catalyze many chemical transformations with reactivity unparalleled by synthetic systems, including reactions relevant to atmospheric health and pollution remediation. Typically such transformations are facilitated by enzyme metal centers that directly interact with small molecules such as nitric oxide, oxygen or methane. A particular challenge in understanding how the enzymes work is the difficulty in characterizing the interaction of the metal center and small molecules. We have developed new spectroscopic methods for characterizing enzymes by directly probing metal centers coordinated to small molecules with synchrotron-produced high intensity X-rays and correlating the results with computational analysis. X-ray absorption spectroscopy (XAS) has been applied to many biological systems, however it cannot distinguish between similar coordination geometries or light atoms bound to the metal center. Our work shows that high energy resolution fluorescence detection (HERFD) XAS and X-ray emission spectroscopy (XES) can distinguish binding modes of nitric oxide at nickel centers, and our experimental spectra have excellent quantitative correlation to computational models. Spectral fingerprints for linear versus side-on bound nitric oxide and neutral versus anionic oxidation states have been determined. These spectral fingerprints establish references for small molecule coordination modes that can be directly applied to complex enzyme intermediates. This is one of the first XES studies of nickel centers and has significant implications for characterizing pollutants bound to enzyme metal centers. The work was supported by Kellogg RSCA, CPP College of Science, SSRL, CSUPERB, and NSF XSEDE.
Poster #: 15
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Cardiovascular disease, Lipoproteins, Oxidative stress
Project Title: Oxidative Stress and Apolipoprotein E in Brain Endothelial Cells
Author List:
Cruz, Siobanth; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Narayanaswami, Vasanthy; Chemistry and Biochemistry, California State University, Long Beach
Abstract: Apolipoprotein E3 (apoE3) is an exchangeable protein that plays a significant role as an anti-atherogenic apolipoprotein. It serves as a ligand for the LDL receptor (LDLr) family of proteins that mediate cellular uptake of lipoproteins and plays a significant role in regulating plasma cholesterol homeostasis. In this study, we investigate the functional consequences of acrolein-mediated oxidative damage to human apoE3 in brain endothelial cells that are the primary constituents of the blood brain barrier. Acrolein is an α,β-unsaturated aldehyde (2-propenal, CH2=CH-CHO) that is considered an environmental pollutant and is also generated endogenously as bye-products of lipid peroxidation. It is considered an oxidative stress reagent that causes indiscriminate oxidative damage to cellular components, including proteins. We test the hypothesis that acrolein modification impairs the structural integrity and functional ability of apoE3. Recombinant human apoE3 was modified with varying concentrations of acrolein, and the modification confirmed by Western Blot analysis using acrolein-lysine-specific antibodies. Modified apoE3 was reconstituted with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and the resultant high density lipoprotein (HDL) was isolated by density gradient ultracentrifugation. Reconstituted HDL bearing acrolein-modified apoE3 does not interact with soluble LDLr in co-immunoprecipitation assays suggesting oxidative modification of essential lysine residues required for interaction with the ligand-binding domain of the LDLr. However, incubation with mouse brain cerebral cortex endothelial cells, showed internalization of acrolein-modified apoE3. Incubation with suramin, an inhibitor of the LDLr, does not abolish cellular uptake of acrolein-modified apoE3. This suggests that alternative mechanisms not involving LDLr are involved in internalization of acrolein-modified apoE3. Studies are in progress to determine the uptake pathway, with emphasis on the class of scavenger receptors as likely candidates. The significance of the study is that it offers insight into the molecular basis of oxidative stress mediated damage to apolipoproteins and their role in pathogenesis of cardiovascular and cerebrovascular diseases.
This project was funded by NIH grant #GM105561
Poster #: 16
Campus: CSU East Bay
Poster Category: Biochemistry
Keywords: Plant stress response, Enzyme, Electrophoresis
Project Title: Two poplar hybrids exposed to salt and boron stress show different polyphenol oxidase isoforms
Author List:
Cuellar, Carlos; Undergraduate, Chemistry and Biochemistry, California State University, East Bay, Presenting Author
Mavi, Prabhjot (Sandy); Undergraduate, Chemistry and Biochemistry, California State University, East Bay, Presenting Author
LeDuc, Danika; Chemistry and Biochemistry, California State University, East Bay
Baňuelos, Gary; Agricultural Research Service – US Department of Agriculture
Sommerhalter, Monika; Chemistry and Biochemistry, California State University, East Bay
Abstract: Agriculture in the Central Valley of California heavily relies on irrigation. Due to high soil salinity, the drainage water contains excess salt and boron. Poplar hybrids of parentage Populus trichocarpa x nigra x deltoids were tested for their ability to tolerate and recycle high salt and boron drainage water. Here we selected two resilient poplar hybrids labeled “Red” and “Blue” and investigated the activity and isoform distribution of the enzyme polyphenol oxidase (PPO). This enzyme is responsible for the oxidative browning in plants and might play a role in plant resilience.
In contrast to most other proteins, PPO is activated and not denatured by the detergent sodium dodecyl sulfate (SDS). We exploited this property and performed in-gel activity staining after SDS polyacrylamide gel electrophoresis (SDS-PAGE) with poplar leaf extracts. Various phenolic substrates (catechol, 3-methylcatechol, 4-methylcatechol, coumaric acid, caffeic acid, chlorogenic acid, L-tyrosine, tyramine, L-DOPA) and buffer conditions were investigated. All SDS-PAGE lanes were loaded with the same amount of total protein as determined via Bradford assays. Leaves were harvested from poplar hybrids irrigated with normal water (control) and with high salt and boron water.
Leaf extracts prepared from the two hybrids showed different staining patterns on SDS-PAGE gels demonstrating that each hybrid contains a unique set of different PPO isoforms. Acidic conditions typically resulted in a single band per hybrid., whereas more PPO isoforms with up to 4 distinct bands were detected in alkaline staining conditions. The substrate 4-methylcatechol yielded the most intense staining patterns. Most surprising was the different stress response of the two poplar hybrids. The “Red” leaves harvested from trees irrigated with high salt and boron water showed overall lower PPO staining intensity compared to control leaves. This was the expected response since lower PPO activity preserves more phenolic compounds, which can then serve as antioxidants. In contrast, the “Blue” leaves, showed more intense PPO staining after high salt and boron irrigation. This same hybrid dependent stress response was also observed in isoelectric focusing (IEF) gels stained for PPO activity. Our next step is to combine IEF and SDS-PAGE in 2D electrophoresis.
This project was funded with a faculty support grant from CSU East Bay.
Poster #: 17
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: Medicinal Chemistry, Metalloprotease Inhibitor , peptidomimetic
Project Title: Sulfonamide peptidomimetic inhibitors for the Botulinum Neurotoxin
Author List:
Cruz, Ricardo; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton, Presenting Author
Amezcua, Martin; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton, Presenting Author
Rodrigues-Beltran, Sandra; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton
Paterson, Brandon; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton
Salzameda, Nicholas; Chemistry & Biochemistry, California State University, Fullerton
Abstract: The Botulinum neurotoxin (BoNT) is one of the most lethal toxins known to man and is secreted by the Clostridium botulinum bacteria. The potency of the toxin and its ease of extraction from the bacteria makes BoNT a threat for use in biological terrorism. BoNTs are composed of heavy chains (HC) and light chains (LC). The LCs are a zinc metalloprotease, which are responsible for cleaving SNARE proteins in the synaptic cleft between neurons and muscles, which cause termination of neurotransmission. This termination results in muscle paralysis that can lead to death. Cleavage of the SNARE protein is irreversible; therefore, treatments are needed to prevent this lethal disease. Current treatments for BoNT intoxication are designed for pre-exposure and are not designed to deal with large infected populations. Our laboratory focuses on synthesizing small molecules that can inhibit BoNT LC as a therapeutic method to treat the disease.
Previously, our laboratory discovered a biphenyl sulfonamide isoleucine hydroxamic acid inhibitor for the BoNT LC. We have expanded on this inhibitor by increasing the number of amino acids in the scaffold while maintaining the biphenyl sulfonyl N-terminal and the hydroxamic acid at the C-terminal. We hypothesized that increasing the number of amino acids would improve binding to the protease. The amino acids that were utilized for this library were alanine, glycine, isoleucine, leucine, phenylalanine and valine. The new inhibitors were created via a solid phase synthetic strategy with hydroxylamine Wang resin. Amino acids were coupled to the resin in sequence and the biphenyl sulfonyl chloride was added to give the sulfonyl-amide bond. The small molecules were cleaved from the resin with trifluoroacetic acid to give the desired molecule in good yield and purity. The order in which the amino acids were coupled was alternated to obtain a library of molecules with sequence diversity. The library of inhibitors was evaluated using an enzymatic assay to measure BoNT inhibition. After examining the peptidomimetic library, it was discovered that three sequences displayed substantial inhibition. The sequences containing alanine-isoleucine, isoleucine-glycine, and isoleucine-valine displayed 89% or greater inhibition at a concentration of 10 µM. These results prove that increasing the overall length and hydrophobic structure to the small molecules improved their binding to the large hydrophobic active site of the BoNT LC.
Poster #: 18
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Phosphorylation, Kinase, Cancer
Project Title: Exploring Phosphorylation as a Mechanism for Regulation of Cyclin-Dependent Kinase 5
Author List:
Roach, Brett; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Tran, Amy; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Bhandari, Deepali; Chemistry and Biochemistry, California State University, Long Beach
Abstract: Protein phosphorylation, a reversible modification, plays a central regulatory role in various cellular processes, such as proliferation, differentiation, migration etc. Thus, over- or under-activation of kinases (the enzymes that carry out phosphorylation) can result in various pathological conditions. Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase whose over-activation/over-expression has been implicated in several pathologies including cancer cell metastasis, diabetes and neuro-degeneration. Therefore, investigating mechanisms that regulate CDK5 activity remains a crucial area of study with far reaching implications in pathological understanding and therapeutics. Here, we have employed in vitro and in cellulo kinase assays to confirm that CDK5 is able to phosphorylate itself. We have identified the potential phosphorylation sites using mass-spectrometry and generated phosphomimetic and non-phosphorylatable mutations at these sites using polymerase chain reaction based site-directed mutagenesis. Phosphomimetic mutation at one of the sites abolished CDK5’s ability to interact with its activator protein suggesting a potential and novel mechanism to keep its activity under check. Our current and future goals include testing the effect of these mutants on cell migration, a key phenotype promoted by CDK5.
This project is supported by the Office of Research and Sponsored Programs grant, CSULB and in part by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R25GM071638.
Poster #: 19
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: DNA Repair, Fluorescence Microscopy, Cloning
Project Title: Construction of a specialized yeast strain for investigating recruitment of the Saw1 and Rad52 proteins in Double Strand Break Repair
Author List:
Vartanian, Lauryn; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Guzman, Jimmy; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Babayan, Melin; Undergraduate, Chemistry and Biochemistry, California State University, Northridge
Fischhaber, Paula; Chemistry and Biochemistry, California State University, Northridge
Abstract: DNA double strand breaks (DSBs) are induced when DNA is exposed to chemical agents or ionizing radiation which in turn cause chromosomal alterations that can lead to cancer in humans. DSBs are repaired by a family of pathways known as the Double Stand Break Repair pathways. Single Strand Annealing (SSA) is one of these pathways that repairs DSBs between two DNA repeats, resulting in complete loss of DNA sequence originally situated between the repeats. In the yeast, S. cerevisiae, Saw1 protein usually recruits the Rad1-Rad10 protein complex to SSA repair sites, possibly by binding to Rad52 protein. Since yeast Rad52 (ScRad52) and human Rad52 (HsRad52) are homologs, we are investigating whether HsRad52 can be recruited to SSA sites in yeast, and if so, whether HsRad52 can recruit yeast Saw1. Toward determining whether HsRad52 can subserve this function of ScRad52, we have constructed a specialized yeast strain containing two inducible DSB sites, each labeled fluorescently with a different color. This strain will enable simultaneous induction of two DSBs on chromosomes V and XV, followed by the monitoring of the annealing step of their SSA repair by fluorescence microscopy during which the two fluorescent labels are brought into close proximity and become colocalized.
Construction of our target strain began by using standard cloning techniques to produce two important fluorescent labeling features: an tandem array of LacO DNA sequences for the LacI DNA binding protein (LacO array) and a LacI gene fused to Yellow Fluorescent Protein (LacI-R197L-YFP). The LacO array was cloned into plasmid pAAH1 and flanked by yeast DNA designed to trigger site-specific integration into chromosome XV, giving plasmid pAAH1-LacO. Likewise, the LacI-R197L-YFP gene was cloned into plasmid pUC57, flanked by yeast DNA designed for integration into the Rad10YFP yeast strain. A progenitor strain containing some specialized genetic elements needed for our fluorescence assay was transformed with pAAH1-LacO. Following transformation, we have screened clones for integration of the Hygromycin selection marker tandemly situated next to the LacO array. Several successful transformants have been identified, one of which will be combined with LacI-R197L-YFP to complete our target strain. Using fluorescence microscopy, the resulting strain will allow us to investigate the formation of colocalized foci in response to induction of DSBs.
The authors thank NIH grant SC3GM093858 for funding.
Poster #: 20
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Galleria mellonella, apolipoprotein, antimicrobial activity
Project Title: Antimicrobial activity of apolipophorin III from Galleria mellonella decreases in lipid-bound form
Author List:
Wijeratne, Tilini; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Weers, Paul; Chemistry and Biochemistry, California State University, Long Beach
Abstract: Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein from the hemolymph of Galleria mellonella. It is extensively used as a model to study lipid transport. ApoLp-III is found in a lipid-free form when the insect is resting. However, during flight activity it associates with diacylglycerol-enriched lipoproteins which are transported from lipid stores to flight muscles. When binding to lipoproteins, apoLp-III undergoes a dramatic conformational change in which the helix bundle opens exposing buried hydrophobic amino acid residues allowing association with the lipid surface. Previous studies have shown that lipid-free apoLp-III binds to lipopolysaccharides (LPS) and phosphatidylglycerol (PG), which are abundant components of the bacterial membrane, potentially acting as an antimicrobial protein. The focus of this project is to study whether apoLp-III retains its antimicrobial activity in the lipid-bound form. Lipid-bound apoLp-III complexes were produced by incubation with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Electron microscopy images of POPC•apoLp-III complexes isolated by ultracentrifugation showed that the particles were disc-shaped with a diameter of 13.6 ± 2.1 nm or 8.6 ± 1.2 nm. The complexes analyzed through non-denaturing PAGE showed two distinct populations of complexes of 458 kDa and 335 kDa, consistent with the electron microscopy images. Protein and lipid content of the discs were determined by the bicinchoninic acid and phospholipid C assay, respectively. This revealed a lipid to protein molar ratio of 40:1 and 10 apoLp-III molecules per disc. Non-denaturing PAGE showed that POPC•apoLp-III complexes were able to disaggregate LPS micelles, however, to a lesser extent than lipid-free protein. The amount of apoLp-III/LPS complexes formed was also less with lipid-bound apoLp-III. Binding to PG membranes was measured by release of fluorescent calcein from PG vesicles induced by apoLp-III. Fluorescence intensity of calcein released by lipid-bound apoLp-III was 51.2 ± 1.2% while lipid-free apoLp-III released 3.6 ± 1.2%. Thus, these results clearly demonstrated that lipid-bound apoLp-III displays a decreased activity to interact with LPS and PG vesicles, indicating that apoLp-III in the lipid-free state has a higher antimicrobial activity compared to lipid-bound apoLp-III. This research was supported by a grant from the National Institutes of Health (NIGMS #GM089564) and the 2016 CSULB Student Summer Research Award.
Poster #: 21
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Bcl-2, metabolism, blood cancer
Project Title: Study of the metabolic effects of Bcl-2 overexpression in a prolymphocyte cell model
Author List:
Abed, Ali; Graduate, Chemistry, California State University, Fresno, Presenting Author
Samaan, Nawras; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Kaur, Preet; Undergraduate, Chemistry, California State University, Fresno
Mahmood, Bushra; Graduate, Chemistry, California State University, Fresno
Dejean, Laurent; Chemistry, California State University, Fresno
Abstract: B- cell lymphoma 2 (Bcl-2) family proteins are the key regulators of the intrinsic pathway in the programmed cell death (apoptosis). Bcl-2 family proteins have both pro-apoptotic and anti-apoptotic proteins. The overexpression of the anti- apoptotic protein Bcl-2 is associated with certain types of diseases such as blood cancer. Our goal is to study Bcl-2 effects on Tumor metabolism. Cancer cells exhibit high rates of lactic acid fermentation compared to normal cells. This has led to the assumption that cancer cells universally shift their metabolism toward anaerobic respiration (i.e. the Warburg effect). However, several studies also indicate that the rate of aerobic respiration is elevated as well in certain types of tumors such as glioma. This suggests that cancer cells may also display elevated rates of energy metabolism in general, rather than an exclusive increase of lactic fermentation.
In our study we used a fetal prolymphocytic murine cell line (FL 5.12) cultivated in an IDMEM 25 mM glucose medium. This cell line has been shown to induce lymphoma when overexpressing Bcl-2 after injection in mice. We also were able to observe that Bcl-2 overexpression leads to an increase in lactic acid fermentation in the same cell line. Basal respiration rates of Parental and Bcl-2-overexpressing FL5.12 cells were measured through measurement of oxygen consumption over time using a Clark-type oxygen electrode (JObasal). Uncoupled (i.e. in the presence of CCCP, JOCCCP) and non-coupled (i.e. in the presence of oligomycin, an inhibitor of the mitochondrial ATP synthase, JOoligo) respiratory rates were also be measured in order to determine the ‘oxidative phosphorylation regime’ of these cells (i.e. [JObasal – JOoligo]/ [JOCCCP – JOoligo]). Our results show no significant change in JObasal, JOCCCP, JOoligo or oxidative phosphorylation regime between or Parental and Bcl-2-overexpressing FL5.12 cells. Taken together, these results indicate that Bcl-2-overexpression driven increase of lactic acid fermentation is not associated to a Warburg-type metabolic shift as respiration rates remained unchanged. Future experiments include the metabolic study of Bcl-2 overexpression in FL5.12 growing in a medium containing 5 mM of glucose only to mimic normal glycaemia.
Poster #: 22
Campus: Humboldt State University
Poster Category: Biochemistry
Keywords: Surface layer protein, bioremediation, bioengineering
Project Title: Evaluation of Lead Peptides Displayed on Caulobacter vibrioides S-Layers Using Fluorescence
Author List:
Coblentz, Azariah C.; Undergraduate, Chemistry, Humboldt State University, Presenting Author
Templeton, Kayla M.; Undergraduate, Chemistry, Humboldt State University, Presenting Author
Cappuccio, Jenny A.; Chemistry, Humboldt State University
Abstract: Heavy metal contamination of soils and waterways due to industrial processes, such as mining, continues to be a problem in the United States. Recent reports indicate that microbial surface layers (S-layers) may be able to bind and sequester heavy metals, thereby removing them from the environment. Here we describe the design and evaluation of lead-binding strains of Caulobacter vibrioides for bioremediation. This organism possesses an S-layer protein (RsaA) which forms a 2D crystalline array with hexagonal symmetry. Because RsaA covers the outermost surface of the cell, specific nanoscale engineered modifications could increase the efficiency for this organism to bind heavy metals. Genetically verified engineered strains were also confirmed to export the S-layer protein as expected. Growth studies in media indicate that recombinant and wild-type strains can tolerate and grow in at least 25 ppm Pb in solution. Using highly sensitive fluorescent dyes, lead remediation is measured in the nanomolar range. Results show that upon exposure to 39.06 nM Pb solution engineered C. vibrioides strains HCm 027,028, remediated the lead remaining in solution to, 9.6 nM ± 290 pM and 8.9 nM ± 190 pM respectively. The wild type strain remediates the lead to 13.3 nM ± 57 pM. These results indicate our engineered strains bind more lead than the wildtype. Future goals of this project include determining association constants of lead to cells, the binding of other heavy metals, and construction of a bioremediation filter.
Poster #: 23
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Flavin Reductase, Protein Purification, Structural enzymology
Project Title: Purification and Characterization of Flavin Reductase, DszD, from Rhodococcus erythropolis
Author List:
Mendez, Gilberto; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Madha, Bilal; Undergraduate, Chemistry and Biochemistry, California State University, Northridge
Vey, Jessica; Chemistry and Biochemistry, California State University, Northridge
Abstract: Flavin reductases are found in all organisms, and catalyze essential reactions in all domains of life. Flavins such as flavin mononucleotide, flavin adenine dinucleotide, and riboflavin are essential for many metabolic processes and are best known for their role in oxidation-reduction reactions. We are working on crystallizing and assessing the catalytic capabilities of the NADH-dependent flavin reductase, DszD, from the biodesulfurizing bacterium Rhodococcus erythropolis. In our work, DszD has been successfully overexpressed and purified essentially to homogeneity from E. coli. We have verified the substrate preferences of DszD, showing the enzyme has maximal activity with flavin mononucleotide. DszD was subjected to a sparse matrix crystallization experiment using the hanging drop vapor diffusion method. Promising crystallization conditions were identified, replicated, and are currently being optimized to improve crystal quality. Once solved, the structure of DszD will lead to the identification of the active site residues important for substrate binding and catalysis. This work was funded by the Research Corporation through CCSA 22672 and the NIH through Grant 5SC2AI109500.
Poster #: 24
Campus: CSU San Marcos
Poster Category: Biochemistry
Keywords: Protein Folding, ribosome, click-chemistry
Project Title: Protein Folding Reactions as Ultrasensitive Environmental Sensors
Author List:
Duarte, Isaac; Undergraduate, Chemistry and Biochemistry, California State University San Marcos, Presenting Author
Huerta, Roxanne; Undergraduate, Chemistry and Biochemistry, California State University San Marcos, Presenting Author
Manrique, Queenie; Undergraduate, Chemistry and Biochemistry, California State University San Marcos
Hamadani, Kambiz; Chemistry and Biochemistry, California State University San Marcos
Abstract: Protein folding is a very sensitive bioconformational reaction that is required for numerous biological functions. The thermodynamics of protein folding reactions is extremely sensitive to perturbation. Here we harnessed this sensitivity to track 1). sample oxidative damage during copper-click bioconjugation reactions, 2). the fidelity of in-vitro translation reactions, and 3). the effects of the co-solvent trifluoroethanol (TFE) on protein folding energy landscapes. For our studies we used green fluorescent protein and hen egg white lysozyme (HEWL) as model systems. Our results support previous findings which indicate that anaerobic copper-click reaction conditions can adequately protect target proteins in bioconjugation reactions from potential oxidative damage. We also find that the folding and maturation efficiencies of fluorescent test proteins synthesized by purified and reconstituted in-vitro translation are far lower than expected. We are investigating the possibility that this may be due to translational infidelity. Finally, using linear extrapolation methods we find that in the presence of small amounts of TFE test proteins are significantly destabilized. While not conclusive, these results support our working hypothesis that in-vitro studies of protein folding reactions in the presence of TFE can recapitulate certain aspects of how the ribosome influences co-translational protein folding reactions.
Poster #: 25
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Bcl-2, metabolism, NMR
Project Title: Cross-talk Regulation Mediated by the Bcl-2 family Proteins between cell survival and Metabolism
Author List:
Llanos, Rhaul; Graduate, Chemistry, California State University, Fresno, Presenting Author
Mahmood, Bushra; Graduate, Chemistry, California State University, Fresno
Krishnan, Krish; Chemistry, California State University, Fresno
Dejean, Laurent; Chemistry, California State University, Fresno
Abstract: Cancer metabolism is characterized by an increase of anaerobic glycolysis vs mitochondrial oxidative phosphorylation to produce energy for cellular processes, (singularities known as the Warburg effect), while The Bcl-2 Family Proteins regulate vital signaling pathways in cancer metabolism; but how these processes are coordinated is poorly understood.
Bcl-2 family proteins contain both pro- and anti-apoptotic members which are respectively encoded by tumor suppressors and proto-oncogenes. Up-regulation of the anti-apoptotic proteins has been associated with Non-Hodgkin’s lymphoma; and previous studies suggest Bcl-2 plays a role in the regulation of energy metabolism by having a key role in the mechanisms related to these metabolic switches which are still a matter of debate.
Our research has focused on molecular regulation of cancer and NMR spectroscopy based metabolomics analysis provided us an approach to study changes in metabolic pathways from Wild type, Bcl-xL, Bcl-2 and Bcl-2-G145E, FL5.12 cell lines.
We monitored the conversion of stable isotope-labeled glucose to look into the metabolic dynamics and tracking of individual carbon routes within carbohydrate metabolism.
The analysis of the stable isotope 13C carbon tracing stable was used to determine the dynamics of metabolic circuitries that are altered due to of Bcl-2 overexpression. Our research helped us to better define an integrated picture of the pathways connecting Bcl-2 and fuel metabolism; and provided clues for new strategies in therapeutics against blood cancer such as Non-Hodgkin’s lymphoma.
These cell lines show significant differences in the profiles of the NMR spectra; one of the affected signals seeming to more specifically reflect changes at the level of the metabolic step catalyzed by the succinate dehydrogenase (i.e. accumulation of fumarate).
Interestingly, we also observed that Bcl-2 or Bcl-xL over-expression led to a significant increase of lactate production rates in a mouse pro-lymphocyte B cell line.
These data suggest that Bcl-2/xL expression levels may play an active role in the regulation of both oxidative metabolism and lactic fermentation (the latter being commonly observed in blood cancer cells); and that this effect may depend on the ability of these anti-apoptotic proteins to physically interact with Bax.
Poster #: 26
Campus: Sonoma State University
Poster Category: Biochemistry
Keywords: persulfides, H2S, Bioinorganic
Project Title: Synthesis and Characterization of Cobalt (II) and (III) Salen and their Possible Interactions with Persulfide Species
Author List:
Holm, Annika ; Undergraduate, Chemistry , Sonoma State University, Presenting Author
Fukuto , Jon; Chemistry , Sonoma State University
Works, Carmen ; Chemistry , Sonoma State University
Abstract: Recent studies indicate that persulfides (RSSH) are highly prevalent in mammalian cells, tissues and plasma. However, their biological/physiological function is, as yet, unknown. Previous work by us alludes to the fact that one of the biological targets for persulfides can be metalloproteins. Thus, the goal of this research project was to determine how persulfides interact with metals/metalloproteins. Persulfide species have been of particular interest because they possess unique chemistry compared to other biological sulfur species. We have proposed that persulfides will readily reduce oxidized metalloproteins. Indeed, among all biologically relevant sulfur species, persulfides are likely the more reducing than thiols. Our long-term goal is to understand the chemistry of persulfides with metalloproteins such as myoglobin. A simple model was used to understand the chemistry of persulfides with stable transition metal complexes. Salen ligand was synthesized, and cobalt (II) acetate was used to make a metal complex. From this complex the cobalt (III) analog was also prepared by oxidation. The cobalt complexes were characterized using UV-Vis spectroscopy and mass spectrometry. A persulfide donor was then used to test for possible metal-persulfide interactions. Surprisingly, Uv-vis results indicate that RSSH oxidizes cobalt (II) but does not react with cobalt (III). Interestedly, Uv-vis results also indicate that RSSH reduce iron(III) in myoglobin. Currently we are working towards understanding mechanisms of these reactions, but conclusions so far indicate that spin states and ligand binding can influence the chemistry of persulfides with transition metals.
Poster #: 27
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: dog copper ceruloplasmin, ,
Project Title: Identification of two additional unusual features of copper transport and metabolism in dogs
Author List:
Kim, Kaitlynne; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Vallabhaneni, Sai; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Flynn, Stephen; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Do, Kathy; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Linder, Maria C; Chemistry and Biochemistry, California State University, Fullerton
Abstract: We have long known that dogs are unusual in the way they handle Cu. Not only are liver concentrations much higher than in other mammals, but their serum albumin has a much lower affinity for Cu. High liver Cu levels are mainly due to a reduced ability to excrete Cu through the bile, so it can accumulate to high levels, causing liver toxicosis. A new mutation in the Cu “pump” Atp7b has recently been implicated in the Cu overload of Labrador retrievers. We have been studying the Cu components of blood plasma and have uncovered additional ways in which dogs differ from most other species.
Blood plasma of Labrador retrievers with/without a hetero or homozygous mutation in Atp7b, were obtained from Dr. Hille Fieten (University of Utrecht); additional samples were from Dr. Scott Weldy (Serrano Animal and Bird Hospital). Whole plasma and ultrafiltrates with components of Canine plasma separated in large pore SEC had a markedly different profile than that seen in other species. The main Cu peak (normally Cp) eluted much earlier, peaking in Fraction 20 rather than 29, suggesting it might be much larger. However Cp protein and enzyme activities also eluted earlier (also for other dog types), indicating canine Cp is either aggregated or binding other proteins. Little or no Cu eluted with albumin, consistent with a lower Cu affinity.
Many Labrador retrievers also had high plasma levels of small Cu carriers (SCCs; These results indicate that the roles of canine Cp and albumin differ from those of other mammals, and suggest that activation of SCC production (due to a particular mutation in Atp7b) may render dogs less susceptible to Cu overload by enhancing loss of excess Cu through the urine.
Poster #: 28
Campus: Sonoma State University
Poster Category: Biochemistry
Keywords: cancer targeted therapy, RNA aptamer, DMS footprinting
Project Title: Synthesis and Characterization of the Tumor Necrosis Factor Receptor BAFF-R in Non-Hodgkin’s Lymphoma
Author List:
Kimberly, Tanner; Undergraduate, Chemistry, Sonoma State University, Presenting Author
Lares, Monica; Chemistry, Sonoma State University
Abstract: B-cell activating factor receptor (BAFF-R) is a protein that belongs to the superfamily of tumor necrosis factor receptors and has been found to regulate B-cell survival. In patients with Non-Hodgkin’s Lymphoma, BAFF-R is overexpressed and thus leads to hyperactive B-cell proliferation and hyperplasia. Previously, a BAFF-R aptamer has been exploited for receptor-mediated endocytosis of medicinal siRNA. This siRNA has proven to be a practical means of inactivating the mRNA of B-cell oncogenes. The objective of our lab is to find a practical means of synthesizing BAFF-R and to characterize its RNA aptamer interaction. We began by using the hallmark method of transforming a BAFF-R plasmid into E. Coli BL21 and then inducing the cells. After the cells had been induced, cell pellets were obtained and then SDS-PAGE was performed. After several procedures, the BAFF-R protein did not appear on the gel. A Western Dot Blot was performed which also gave further evidence to support the absence of BAFF-R; this led us to believe that BAFF-R may be toxic to the E. Coli cells. A toxicity test was conducted by isolating the DNA of induced and non-induced cells and comparing the concentrations by restriction digest and agarose gel. After running the agarose gel, it was clear that the induced cells had much less plasmid than the non-induced cells – an indication that the viability of the induced cells had been compromised. Due to the fact that E. Coli cells are insufficient for the expression of BAFF-R, our lab is investigating alternative methods of protein synthesis such as the use of insect cells or in vitro synthesis. One of these alternative methods may lead to a successful synthesis of BAFF-R which will in turn allow for the characterization of the BAFF-R and RNA aptamer interaction.
Poster #: 29
Campus: CSU East Bay
Poster Category: Biochemistry
Keywords: Tritonia diomedea, enzyme, neurotransmission
Project Title: Molecular and kinetic properties of acetylcholinesterase isoforms in Tritonia diomedea
Author List:
Makani, Leena Priya ; Graduate, Chemistry and Biochemistry, California State University, East Bay, Presenting Author
Tran, Vu; Graduate, Chemistry and Biochemistry, California State University, East Bay
Murray, James; Biology, California State University, East Bay
Sommerhalter, Monika; Chemistry and Biochemistry, California State University, East Bay
Abstract: Acetylcholinesterase (AChE) plays an important role in the nervous system, terminating impulse transmissions at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine. The marine gastropod Tritonia diomedea (a.k.a. T. tetraquetra) serves as a model organism in neurobiology. We discovered at least four different AChE isoforms in the nervous system of T. diomedea. By characterizing these different AChE isoforms we aim for a better understanding of cholinergic signaling in this invertebrate model organism.
We separated cytosolic and membrane associated AChE isoforms from brain and buccal ganglia. Specific AChE activity was highest in the cytosolic buccal ganglia extract reaching up to 4 U/mg protein and lowest in the cytosolic brain tissue extract yielding less than 0.2 U/mg protein. The samples from the nervous system showed similar Michaelis-Menten constants ranging from 0.15 to 0.4 mM, but differed in their substrate inhibition constants ranging from 5 to 64 mM. Notably, the detergent soluble buccal ganglia sample showed no substrate inhibition. The physiological implications are intriguing. Substrate inhibition modulates the time course of cholinergic signals. At the on-set of the cholinergic signal more neurotransmitter molecules remain at the synaptic cleft as their enzymatic degradation is attenuated. At signal termination the AChE activity increases as substrate concentration diminishes. The membrane associated AChE isoform in the buccal ganglia hence does not attenuate neurotransmitter degradation at the cholinergic on-set.
To complement this data set we aim to determine the apparent molecular weight and oligomerization state of the AChE isoforms. We tested two different staining protocols for native polyacrylamide gel electrophoresis using commercially purified AChE from electric eel. The traditional AChE staining method developed by Karnowsky and Roots with acetylthiocholine, copper sulfate, and ferricyanide required at least 10 micrograms of purified enzyme. In contrast, an optimized cholinesterase staining procedure based on alpha-naphthylacetate and fast blue BB salt required as little as 0.1 microgram. Our most current trial with AChE containing hemolymph showed that further sample optimization is necessary to avoid band smearing with unpurified samples.
This project was funded by a Faculty Support Grant for Collaborative Research from CSU East Bay.
Poster #: 30
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Listeria monocytogenes, Single-domain antibody, Fluorescence microscopy
Project Title: Single-Domain Antibody as an Important Antagonist to Inhibit Listeria Infection
Author List:
Toride, Moeko; Graduate, Chemistry, California State University, Fresno, Presenting Author
Mendoza, Matthew; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Brooks, Cory; Chemistry, California State University, Fresno
Abstract: Listeria monocytogenes (Listeria) is a foodborne pathogen that can induce a life-threatening infection called listeriosis. The infection is particularly harmful during pregnancy as the bacteria can penetrate the placental barrier and infect the developing fetus leading to abortion. The bacteria express a surface protein, Internalin B (InlB), which facilitates bacterial invasion into mammalian host cells. InlB is a multi-domain protein that specifically binds the hepatocyte growth factor receptor, c-MET. Interestingly, the Leucine-Rich Repeat (LRR) domain of InlB constitutes a concave surface which is ideal for receptor-ligand interactions and thus is a suitable target to negate the pathogenicity of Listeria.
The single-domain antibody (VHH) are antibody fragments derived from the heavy-chain antibody found in camelids. The small size of the VHH, high affinity, and specificity are intriguing prophylactic characteristics of these antibodies. We have isolated several VHHs that bind to the LRR domain of InlB with high affinity. Therefore, the objective of this research is to validate the hypothesis that InlB specific VHH can neutralize Listeria invasion.
The effectiveness of VHH for prevention of Listeria invasion was examined using microscopy. Green fluorescence protein (GFP) expressing Listeria were generated by transforming a plasmid that encodes for GFP into Listeria cells. The GFP-Listeria was biotinylated and incubated with HeLa cell cultures in the presence and absence of VHH. The extracellular and attached GFP-Listeria was stained red with streptavidin protein, DyLight 550 conjugate. The sample was analyzed under a Leica DMI3000 B fluorescence microscope.
The HeLa cells infected with GFP-Listeria in the presence of VHH showed that red Listeria cells as many as green Listeria. In contrast, HeLa cells infected with GFP-Listeria in the absence of VHH showed only few red Listeria cells while many green Listeria cells were observed. In conclusion, the fluorescence microscopy experiment demonstrated that VHH successfully inhibits Listeria invasion into HeLa cells.
The project is funded by the National Institutes of Health (grant #: 1SC3GM112532-02).
Poster #: 31
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: West Nile Virus, Medicinal Chemistry , protease inhibitor
Project Title: Synthesis and Evaluation of Small Molecule Inhibitors for the West Nile Virus NS2B-NS3 protease
Author List:
Martinez, Anastasia; Graduate, Chemistry & Biochemistry, California State University, Fullerton, Presenting Author
McBee, Shannon; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton, Presenting Author
Salzameda, Nicholas; Chemistry & Biochemistry, California State University, Fullerton
Abstract: Flaviviruses are neuroinvasive, disease-causing viruses that are transmitted to humans via mosquitoes. They pose a serious threat to human health and are responsible for numerous fatalities each year. West Nile Virus (WNV) is a prominent species of flavivirus and even though most cases are mildly symptomatic, a significant number can progress to detrimental neurological diseases. Currently, there are no vaccines or antiviral therapeutics for prevention and treatment of WNV infections. However, there is ongoing research to discover an inhibitor for the NS2B-NS3 protease, as a promising therapeutic target.
The NS2B-NS3 protease is responsible for cleaving the viral polyprotein at multiple sites to produce the components necessary for the virus to infect and replicate in the host cell. Therefore, inhibition of this serine protease would essentially halt viral replication and discontinue the spread of the virus in the body.
Our laboratory has screened chemical libraries by means of a high throughput enzyme assay to identify inhibitors for the WNV NS2B-NS3 protease. An FDA approved therapeutic drug for the treatment of chronic asthma was identified as an inhibitor for the NS2B-NS3 protease. By performing inhibition studies with detergents at various concentrations, it was validated as a true inhibitor with a competitive mode of inhibition. Through a short structure activity relationship study, we explored the structural appendages of the compound for inhibitor activity and identified the 5-nitroindole to be the active core for inhibition of the NS2B-NS3 protease.
Through short synthetic schemes, we built upon the 5-nitroindole to analyze the effect of placing substituents on the nitrogen in the ring of the indole on inhibition. These substituents were varied by chain length, incorporation of benzenes, and other functional groups. These compounds and their subsequence evaluation by the enzymatic assay reveled that the 5-nitro-N-methylated indole connected to a phenyl ring via a sulfonamide linkage exhibited good inhibition for the WNV NS2B-NS3 protease. It was also determined that the position of the nitro group on the indole is critical for inhibition. With this knowledge new inhibitors are being created that exploit these structural requirements.
Poster #: 32
Campus: San José State University
Poster Category: Biochemistry
Keywords: methylotrophy, lanthanide, methanol oxidation
Project Title: Identification and Characterization of Two Novel Genes Involved in Lanthanide-dependent Methanol Oxidation
Author List:
Chrisostomo, Ralph; Graduate, Biological Sciences, San José State University, Presenting Author
Yarza, Fauna; Undergraduate, Biological Sciences, San José State University
Velosa, Isabel; Undergraduate, Microbiology and Molecular Genetics
Kahn, Nabila; Undergraduate, Microbiology and Molecular Genetics, San José State University
Martinez-Gomez, Norma; Microbiology and Molecular Genetics
Skovran, Elizabeth; Biological Sciences, San José State University
Abstract: Methylobacterium extorquens is a model organism for understanding methylotrophic growth, which is of particular interest due to its potential to produce value added chemicals such as bioplastics and biofuels from methanol. During the first step of methanol metabolism, methanol is oxidized to formaldehyde by methanol dehydrogenase (MeDH). Recently it was discovered that some methylotrophs have the unique ability to oxidize methanol using lanthanides and studies have shown that M. extorquens has a PQQ- and lanthanide-dependent MeDH, XoxF. Lanthanides, also known as rare-earth elements, are of high commercial value for their importance in a wide range of our technologies, from computers to transportation. Mining for lanthanides requires harsh extraction methods that are harmful to the environment. Because M. extorquens is able to acquire insoluble lanthanides from the environment, we can engineer this organism as an environmentally friendly platform for the recovery of lanthanides from end-of-life products and mining ores. Currently, very little is known about lanthanide-dependent metabolism. Transposon mutagenesis identified two genes of unknown function as important for lanthanide-dependent growth, META1_1746 and META1_1747. To determine the roles of these genes in methanol oxidation, null mutations were constructed in each gene and the mutant strains were assed for growth. Loss of either gene resulted in a growth defect that was similar to disrupting the xoxF gene itself. XoxF was purified from wild-type and 1746 and 1747 mutant strains. Intriguingly, though expression of xoxF was high, XoxF protein yields were significantly lower when purified from the mutants. This could suggest that XoxF cannot fold properly in the mutant strains and precipitates out of solution. Of the protein that was purified from the 1746 and 1747 mutant strains, the specific activity was not detectible and 0.02U/mg respectively compared to 5.0 U/mg when purified from the wild-type strain. Future studies will analyze lanthanide content of the purified XoxF-MeDH using inductively coupled plasma mass spectroscopy and PQQ content using visible spectroscopy. This work will further our understanding of the requirements for functional XoxF-MEDH protein and further our engineering efforts in the development of a lanthanide recovery platform. Funding for this project was provided by San José State University and CSUPERB.
Poster #: 33
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: butyrylcholinesterase, amino acids, enzymology
Project Title: Identifying Protected Amino Acids as a Class of Potent and Selective Butyrylcholinesterase Inhibitors
Author List:
Ramirez, Jennifer; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Gonzalez, Jeannette; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach
Schwans, Jason; Chemistry and Biochemistry, California State University, Long Beach
Abstract: Disruption of cholinesterase activity has been implicated in neurodegenerative diseases such as Alzheimer’s. While acetylcholinesterase (AChE) activity decreases or remains unchanged, butyrylcholinesterase (BChE) activity increases in Alzheimer’s patients. Multiple classes of BChE-specific inhibitors have been evaluated as potential therapeutics to manage the progression of neurodegenerative diseases, and previous studies identified molecules bearing aromatic and carbamate groups as potent inhibitors. We evaluated amino acids bearing the 9-fluorenylmethyloxycarbonyl (Fmoc) group, as Fmoc-amino acids contain aromatic and carbamate groups. We determined the effect of Fmoc-amino acids on BChE activity via steady-state kinetics experiments monitored by UV-Vis spectroscopy and identified Fmoc-Leu-O-, Fmoc-Trp-O-, and Fmoc-Lys-O- as the most effective inhibitors. The inhibition constants (KI values) for the analogs were 115, 150, 193 μM for Fmoc-Leu-O-, Fmoc-Lys-O-, and Fmoc-Trp-O-, respectively, values comparable to inhibition constants reported for other classes of inhibitors. Experiments with AChE showed the analogs did not affect activity, suggesting the compounds are specific for BChE. To investigate the physical basis for cholinesterase selectivity, we calculated the van der Waals volumes of the AChE and BChE active site gorges and the Fmoc-amino acids. Our results suggest the smaller AChE active site gorge is unable to accommodate the Fmoc-amino acids but the larger BChE gorge can accommodate the compounds, thereby providing selectivity. Experiments with carboxybenzyl (Cbz) amino acids and side-chain modified amino acids suggested the Fmoc group is important for inhibition and identified Fmoc-Trp bearing a t-butoxycarbonyl on the indole side chain (Fmoc-Trp(Boc)-O-) as the most potent inhibitor with a KI value of 20 μM. Together, the results identify Fmoc-amino acids as potent and selective BChE inhibitors. As Fmoc-amino acids are readily synthesized, we are currently investigating if additional analogs and short peptides bearing Fmoc-amino acids are potent and specific BChE inhibitors.
This project was supported by the National Institute of General Medical Sciences, National Institutes of Health (NIH) under award numbers T34GM008074, UL1GM118979, and RL5GM118978, and the National Science Foundation (NSF; MRI CHE-1337559). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or NSF.
Poster #: 34
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: Paralogs, RNA Binding Proteins, Post-Translational Modifications
Project Title: Understanding the role of post-translational modifications on the splicing activity of two related RNA binding proteins
Author List:
Reynaga, Janice ; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Marshall, Collin; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Keppetipola, Niroshika; Chemistry and Biochemistry, California State University, Fullerton
Abstract: Several human diseases including cancer and Alzheimer’s disease are associated with alternative splicing, one mechanism by which a diverse amount of proteins are produced from a single gene in eukaryotic cells. Alternative splicing is regulated in part by RNA Binding Proteins. The Polypyrimidine Tract Binding Protein (PTBP) is an RNA binding protein that has many functions including alternative splicing regulation and mRNA localization. The PTBP gene family has 3 paralogs; PTBP1, PTBP2 and PTBP3. The paralogs have high primary structure identity and similar domain organization yet has tissue specific expression patterns and different splicing effects on certain target exons. In our study, we focus on related proteins PTBP1 and PTBP2. PTBP1 is expressed near ubiquitously, however is absent in neurons and muscle cells. PTBP2 is expressed in neurons. The two proteins have 74% primary structure identity however exert different splicing outcomes on certain regulated exons. The levels of the two proteins change during neuronal differentiation and maturation and this change in protein concentration is critical for neuronal development. Recent studies indicate that multiple determinants over the regions of the two proteins dictate their differential splicing activity. Genome-wide mass spectrometry studies indicate that the two proteins are post-translationally modified. The hypothesis underlying our studies is that the modifications dictate the differential splicing activity of the two proteins. To test this, we have overexpressed PTBP1 and PTBP2 in HEK293T and mouse N2A cells and assayed via Western Blot. We have probed for protein phosphorylation via Phos-Tag Gel Electrophoresis. We have probed for ubiquitiantion and acetylation using modification specific antibodies via Western Blot. We have purified Flag-tagged PTBP1 and PTBP2 via Flag-Immunoprecipitation for mass spectrometry analysis for post-translational modifications. Preliminary results indicate that the proteins are modified in HEK293T, N2A cells and that they have different interacting partner proteins.
Poster #: 35
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: NMR , Spider Silk, Cryo-EM
Project Title: Probing Silk Protein Oligomeric Assemblies in Spider Silk Glands with NMR Diffusion Measuements and Cryo-EM
Author List:
Onofrei, David ; Graduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Larson, Tanor ; Undergraduate, Department of Chemistry and Biochemistry, San Diego State University
Villalba, Alex; Undergraduate, Department of Chemistry and Biochemistry, San Diego State University
Holland, Gregory; Department of Chemistry and Biochemistry, San Diego State University
Abstract: As biotechnology moves towards renewable and environmentally friendly materials, biopolymers have emerged as promising replacements for the petrochemical-based polymers commonly used to date. Spider silks are a broad category of biopolymers that have a multitude of useful properties including strengths that surpass most man-made materials. We have applied a host of experiments to probe the molecular origins of these properties and have already made strides in understanding the secondary structural contributions to silk fibers using solid-state nuclear magnetic resonance (NMR) and other techniques. However, the mechanisms behind the actual formation of these fibers remains a mystery. Silk fibers are synthesized and stored in specialized glands until they are sent to the spinneret via a duct where the solvated protein fibrils begin to nucleate and finally are spun as solid fibers essentially on demand. In our most recent work, we have focused on silk protein diffusion measurements with NMR, and high-resolution imaging using cyro-electron microscopy (cryo-EM) on the silk protein spinning solution. From the combination of these two techniques, we have begun to form a working model of the silk protein structure, dynamics and organization in the silk-producing gland. We have determined the diffusion coefficient of black widow silk proteins in the gland to be on the order of 2×10-9 cm2/s indicating the presence of large oligomeric silk protein assemblies. By treating the silk protein solution with a denaturant such as urea, the silk protein oligomers could be broken up as confirmed by an increasing diffusion coefficient as a function of time in urea. We correlated this to the possibility of protein micelle-like structures where the hydrophobic core regions of silk proteins are buried and only the polar terminal regions are exposed to solvent. Preliminary data from cryo-EM illustrates the presence of spherical globular structures on the order of 200 nm in diameter and very well could be the first images of these silk protein assemblies. Lastly, dynamic light scattering (DLS) is being applied to a dilute solution of these structures to give complimentary information on silk protein oligomer size and morphology. Based on this hypothesis, spider silk formation could conceivably be controlled by modulating the conditions that lead to the formation of these protein micelles that are expected to be the site of biopolymer fiber nucleation.
Poster #: 36
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: apolipoprotein, apoA-I, lipopolysaccharides
Project Title: The importance of lysine residues of the C-terminal domain of apolipoprotein A-I to for the binding to lipopolysaccharides
Author List:
Voong, Calvin; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Weers, Paul; Chemistry and Biochemistry, California State University, Long Beach
Abstract: Human apolipoprotein A-I (apoA-I) is the major protein in high density lipoprotein, which plays an important role in facilitating reverse cholesterol transport from peripheral tissues to the liver for processing. ApoA-I is a 243 amino acid residue, amphipathic ɑ-helical protein that consists of two domains: an N-terminal helix bundle domain and a less structured C-terminal (CT) domain, which possesses a high lipid binding affinity. In recent years, studies have shown that apoA-I exhibits antimicrobial properties, in which positively charged lysine residues bind to negatively charged membrane components of gram-negative bacteria, such as lipopolysaccharides (LPS) and phosphatidylglycerol (PG). LPS has been known to induce sepsis and septic shock, a condition in which the endotoxic lipid A portion induces an overreaction of the immune system, leading to organ damage and eventually death. ApoA-I contains 15 positively charged lysine residues, which may play an important role in binding and neutralizing negatively charged LPS and PG. In this study, the binding interaction between the lysine residues of the CT domain of apoA-I and LPS will be investigated. The 6 positively charged lysine residues of CT-apoA-I were mutated to glutamine (CT-apoA-I 6KQ), a polar uncharged amino acid. CT-apoA-I 6KQ binding to LPS was studied by non-denaturing PAGE and tryptophan fluorescence. Non-denaturing PAGE analysis showed that the binding interaction of CT-apoA-I 6KQ with LPS was reduced compared to wild-type apoA-I. Tryptophan fluorescence intensity of wild-type apoA-I increased upon LPS addition, indicating an altered tryptophan environment resulting from a binding interaction between apoA-I and LPS. However, the fluorescence emission intensity of CT-apoA-I 6KQ remained unchanged upon LPS addition. These results indicate that the ability of the lysine mutant to bind to LPS was diminished and that lysine residues of CT-apoA-I are important in binding to gram-negative bacterial membranes. To better understand the interaction between wild-type and CT-apoA-I 6KQ with the bacterial membrane, future studies will include investigation of the binding interaction of apoA-I with PG vesicles. This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number SC3GM089684 and R25GM071638.
Poster #: 37
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Lipoprotein, Chimera, Protein structure
Project Title: Design of an apolipophorin-III/apolipoprotein E C-terminal domain chimera
Author List:
Kakutani, Leesa; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Horn, James; Graduate, Chemistry and Biochemistry, California State University, Long Beach
Weers, Paul; Chemistry and Biochemistry, California State University, Long Beach
Narayanaswami, Vasanthy; Chemistry and Biochemistry, California State University, Long Beach
Abstract: Human apolipoprotein E (apoE) is a two-domain anti-atherogenic lipid transport protein that mediates plasma cholesterol homeostasis by serving as a ligand for the low density lipoprotein (LDL) receptor. Of its three isoforms E3, E2, E4, the latter two have been associated with increased risk of cardiovascular and Alzheimer’s disease respectively, yet all three contain an identical C-terminal (CT) domain. The CT domain (residues 201-299) is responsible for tetramerization and has been found to initiate lipid binding that influences critical functional features of apoE. The N-terminal (NT) domain of apoE has four α-helices arranged in a bundle similar to apolipophorin III (apoLp-III), a model insect apolipoprotein containing five α-helices in one domain. To better understand the role the apolipoprotein domains play in structure and function, a novel chimeric apolipoprotein was designed by attaching apoE-CT to apoLp-III. A disulfide bond was introduced to apoLp-III to lock the helix bundle and eliminate its lipid binding. Recombinant apoLp-III/apoE-CT, apoE3, apoE-CT, and apoLp-III were expressed in bacterial cells, purified by affinity chromatography, and the purity verified by SDS-PAGE. Western blot analysis using monoclonal apoE-CT specific antibody confirmed the presence of apoE-CT in the chimera. Crosslinking studies using dimethylsuberimidate revealed that the apoLp-III/apoE-CT chimera formed oligomers similar to apoE, while apoLp-III was monomeric. Far UV circular dichroism showed increased α-helical content, and incubation with 1-anilinonaphthalene-8-sulfonic acid, an environmentally-sensitive amphiphilic dye, showed increased fluorescence intensity when apoE-CT was added to apoLp-III, consistent with the presence of water-shielded dye binding sites upon addition of a structured apoE-CT segment. Our results suggest that the chimeric protein has structural features similar to the parent proteins, and that self-association properties of apoE can be transferred to apoLp-III. Future studies will include measuring the ability of the chimera to solubilize phospholipid bilayers and binding to low density lipoproteins. The chimeric approach offers the potential to obtain insight into domain interactions and the structure-function relationships in apolipoproteins.
This research was supported by the National Institute of General Medical Sciences of the NIH (8UL1GM118979-02, 8TL4GM118980-02, 8RL5GM118978-02, GM089564 and GM105561).
Poster #: 38
Campus: CSU Bakersfield
Poster Category: Biochemistry
Keywords: Coffee, Cancer, Phytochemicals
Project Title: The Impact of the Degree of Roast Levels of Coffee Extracts on its Possible Anticancer Activity
Author List:
Mojica, Benigno; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield, Presenting Author
Forester, Sarah; Chemistry and Biochemistry, California State University, Bakersfield
Abstract: According to the American Cancer Society, colon cancer is the third most common type of cancer in the United States for both men and women. Coffee is one of the most widely consumed beverages in the world and is regarded for its stimulating and anticancer effects. Brewed coffee contains numerous phytochemicals that are beneficial to consumer health. Of those phytochemicals found in coffee, some are known antioxidants with anticancer qualities. The amount of total beneficial compounds present in coffee can be affected at any point from the harvest to the actual preparation of the beverage. In this study, we compared the anticancer activities various freeze dried coffee beverages, differing only in their roast treatments. The different roasting stages selected for this study were green, “Cinnamon”/Blonde, “City”/Medium, “Full City”/Medium-Dark, and “Full City Plus”/Dark. The freeze dried coffee samples were re-suspended in cell culture media and incubated with human HT-29 colon cancer cells at various dilutions of the original brew concentrations. After 72 h of treatment, cell viability was quantified. The green coffee treatment had the least potent growth inhibitory properties followed by the cinnamon coffee. The City, full city, and full city plus coffees all had similar growth inhibitory properties with respect to anticancer potency, and were found to reduce colon cancer cell viability to a greater extent compared to the cinnamon or green coffee treatments. In conclusion, the consumption of coffee at medium to full roast, along with a healthy and balanced diet, may contribute to the prevention of colon cancer.
Poster #: 39
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: terpyridine, DNA, cancer
Project Title: DNA binding by terpyridine based anti-tumor candidates and their complexes
Author List:
Ward, Ashley; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
De Jeronimo Diaz, Cesar; Undergraduate, Kinesiology, California State University, Northridge, Presenting Author
Kelson, Eric; Chemistry and Biochemistry, California State University, Northridge
Abstract: 2,2’:6’,2”-Terpyridines (Terpys) have been demonstrated as promising anti-tumor agents that interfere with DNA duplication and transcription by intercalation (inserting themselves into the base-pair stack) much like daunorubicin. Interestingly, Terpys also exhibit selectivity for tumor cells over normal ones likely by exploiting a specific means of uptake. Despite this promise, Terpys are largely unexplored due to difficulties in preparing them. We have developed methodologies for conveniently making Terpys (and their ruthenium complexes) with appendages designed to intercalate DNA. The purpose of this work is to survey these new compounds for their binding to DNA and deduce the features that give the best affinity. A battery of techniques were employed to survey the strength and nature of binding to DNA. First, DNA binding was confirmed by changes in the UV-visible spectrum of the agent as it was titrated with DNA. The relative strength of DNA binding was probed through attenuation of ethidium bromide fluorescence as it was competitively displaced from DNA during agent addition. To distinguish DNA binding by intercalation, the viscosity of DNA solutions was measured for increases with agent binding. Further, minor grove binding was assessed through the attenuation of Hoechst Blue fluorescence as it was competitively displaced from DNA by agent binding. Terpy itself was confirmed as a good DNA intercalator as a control. Surprisingly NapTerpy (with a 1-naphthyl group) only modestly bound DNA but still appeared to intercalate through the major grove. PhanTerpy (bearing a 9-phenanthryl group) appeared to have a greater affinity for DNA. Ruthenium complexes of Terpy and TerpyTerpy were good binders of DNA, but they appeared to bind electrostatically (in the major grove) rather than intercalating. This indicated that metal bound and pendant (unbound) Terpy groups did not intercalate like free Terpy. Surprisingly, ruthenium complexes of NapTerpy were poor binders of DNA suggesting that the Nap group was too small for stable intercalation. However, ruthenium complexes of PhanTerpy were good DNA binders though they intercalated via the minor grove. Overall, substitution of Terpy inhibits its own intercalation into DNA, but large polyaromatic appendages restore this ability in the free ligands and their complexes. We thank CSUPERB (Research Development Grant) and the NIH (SCORE S06 GM48680, GM063787, 8TL4GM8977-02, and 5RL5MD009603-02) for support.
Poster #: 40
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: heat-shock proteins, lipids, plasma membrane
Project Title: Interaction between HspA1A, a 70-kDA molecular chaperone, and phosphatidylserine in stressed human cells
Author List:
Bilog, Andrei; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Labanieh, Cedra; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Nikolaidis, Nikolas; Biological Science, California State University, Fullerton
Abstract: Seventy-kilodalton heat shock proteins (Hsp70s) are molecular chaperones essential for maintaining cellular homeostasis and survival. Apart from their indispensable roles in protein homeostasis specific Hsp70s also localize at the plasma membrane and bind to several lipids, including phosphatidylserine (PS). The interaction of Hsp70s with PS has direct physiological outcomes including activation of the immune system, microautophagy, and regulation of cell death. However, the conditions that trigger and promote the interaction between HspA1A and intracellularly localized PS in human cells remain unknown. To shed light to this newly described property of HspA1A, we tested whether heat-shock, a stress known to affect both HspA1A expression and membrane lipid composition, triggers the binding of the chaperone to PS. To test this prediction, we first determined whether HspA1A and the C2 domain of lactadherin (Lact-C2), a known PS-biosensor, compete for binding to intracellular PS. Specifically, HeLa cells were subjected to mild heat-shock and the competition was assessed by quantifying the amount of membrane localized HspA1A in the presence or absence of the Lact-C2 protein. These imaging experiments revealed that in the absence of Lact-C2 HspA1A’s membrane localization increases continuously after heat-shock and reaches a maximal value at 8 hours during recovery. In the presence of Lact-C2, however, HspA1A’s membrane localization was minimal and did not show an increase during recovery after heat-shock. These experiments were verified using cell surface biotinylation. In these experiments, HeLa cells, after heat-shock were incubated with membrane impermeable biotin, lysed, and incubated with streptavidin agarose beads, and the biotinylated proteins were analyzed by western blots and quantified using densitometry. Additional imaging experiments revealed colocalization between HspA1A and fluorescently labeled PS (Topfluor-PS). For these experiments co-localization was determined based on the Manders’ overlap correlation coefficient. Collectively, these results strongly suggest that HspA1A bind to intracellular PS, and that HspA1A’s membrane localization and anchorage depends on its interaction with PS. This discovery institutes PS as a new and dynamic partner in the cellular stress response and establish the required foundation to directly assess the biological implications of this newly described and largely uncharacterized function of Hsp70s.
Poster #: 41
Campus: CSU Fullerton
Poster Category: Bioengineering
Keywords: gait rehabilitation, orthotics, mechanism design
Project Title: Design of an Orthotic Device for Restoration of Normal Physiological Gait
Author List:
Shehab, Ahmed ; Undergraduate, Mechanical Engineering, California State University, Fullerton, Presenting Author
Al Hazza, Abdulsahib; Undergraduate, Mechanical Engineering, California State University, Fullerton, Presenting Author
Rocha, Hilen ; Undergraduate, Mechanical Engineering, California State University, Fullerton
Alkharas, Abdullaziz; Undergraduate, Mechanical Engineering, California State University, Fullerton
Ghosh, Shramana; University of California, Irvine, Presenting Author
Robson, Nina; Mechanical Engineering, California State University, Fullerton
Abstract: Restoration of mobility following stroke or spinal cord injuries is a major task in neuro-rehabilitation. Modern concepts of motor learning favor intense task-specific repetitive gait therapy. While the latter has shown great promise, this method has many disadvantages: physical effort required by multiple therapists, achievement of consistent repeatability, cost of treatment, availability and length of training. To alleviate these issues, during the last two decades, a number of robotic orthotics, mobility aids and exoskeletons have been designed.
The current project hypothesis is that developing of a multi-axis knee mechanism will provide a more natural hip movement and lead to increased comfort for users compared to the traditionally used single axis knee design orthotics. In relation to that, a prototype of a wearable walking device incorporating a four-bar multi-axes knee was designed taking into account contact and curvature specifications between the foot and the ground in vicinity of the two key positions: toe-off and heel- strike. The tests showed that the hip height of subjects wearing the device lifts up by 2.1% as compared to a commercially available device with a single axis knee joint. At the same time, while using the device, the hip height sags by 7.6 % compared to humans’ natural gait. Increased stride length with 11.8% as compared to the single-axis knee device was also observed. The stride length of the developed wearable device was short by 19.1% compared to normal walking. Based on these results, it was concluded that the inclusion of passive knee mechanism is not sufficient for obtaining natural walking gait.
As a next step, a new wearable orthotic device that guides the knee, ankle and foot along a natural walking trajectory was developed, based on motion capture data for a healthy human subject walking at 0.5 km/hr on a treadmill. An eight-bar Stephenson II function generator was designed to coordinate the angular motion of the knee and the ankle from the data collected. Of the 332 obtained design candidates, the most compact solution was used for the development of the new wearable orthotic device. The device was deployed in parallel to the human lower extremity and showed promising results of enabling repetitive practice of gait-like patterns. Future directions include further testing and assessment of the performance of the physical prototype, using motion capture system, force-plates and electromyogram (EMG) studies.
Poster #: 42
Campus: CSU San Bernardino
Poster Category: Bioengineering
Keywords: metal, reactive oxygen species, sensors
Project Title: Using Drosophila Reporters to Evaluate Metal Uptake, ROS Production, and Chelation in Drosophila S2 Cells and Human HeLa and Neuroblastoma Cells
Author List:
Fejzic, Hannah; Undergraduate, Chemistry and Biochemistry, California State University, San Bernardino, Presenting Author
Gallardo, Jennifer ; Undergraduate, Biology, California State University, San Bernardino
Garcia, Kayla; Undergraduate, Chemistry and Biochemistry, California State University, San Bernardino
Peresuh, Simbarashe; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Rager, Kaythryn; Undergraduate, Chemistry and Biochemistry, California State University, San Bernardino
Talavera, Estevan; Undergraduate, Chemistry and Biochemistry, California State University, San Bernardino
Abstract: The goal of our project is to determine the rates of metal uptake and reactive oxygen species (ROS) production in Drosophila S2 cells and human HeLa and neuroblastoma cells using a Drosophila metal- and ROS-sensitive luciferase reporter. An additional goal was to use these reporters to examine the effects of metal chelation in ROS production in these cells since metal chelators are suggested as potential therapeutics for neurodegenerative diseases. First, we performed time course experiments to estimate the rates of metal uptake and ROS production in S2 cells by measuring the luciferase signals from the Mtn and SOD promoters after treating the cells with Cu, Fe, and Zn for different time periods. From this study, we found that the luciferase signal from the Mtn promoter construct increased linearly over time when the cells were treated with Cu and Zn, but not with Fe. This trend was also observed with the SOD promoter construct. Together these results indicate a correlation between increased Cu and Zn uptake and increased ROS levels in S2 cells. For our chelation tests, we used EDTA, citrate, and histidine because they have been suggested as potential therapeutics for neurodegenerative diseases. We performed the chelation experiments in Drosophila S2 cells and human HeLa and SHSY cells by measuring the luciferase activities from the Mtn and SOD promoters in cells treated either with Cu, Fe, or Zn in the presence or absence of EDTA, histidine, or citrate. Of the nine metal-chelator combinations tested in S2 cells, the only combination that led to a statistically significant decrease in Mtn-luciferase activity was Zn and citrate (p< 0.10), which correlated with a corresponding decrease in SOD-luciferase activity. Our results suggest that citrate is able to chelate Zn, thus reducing its ability to generate ROS. The same effect was observed in HeLa and SHSY-5Y cells with the Mtn promoter construct. In addition to this, we found that in HeLa cells treated with Cu and histidine, there was a 2-fold decrease in Mtn-luciferase activity relative to cells treated with histidine alone. There was also a significant decrease in Mtn-luciferase activity in SHSY-5Y cells treated with Fe and all three chelators relative to cells treated with Fe alone.
This project was supported by the CSUSB PRISM Program (NSF DMS-1035120).
Poster #: 43
Campus: CSU Fullerton
Poster Category: Bioengineering
Keywords: Orthotic Arm, Electromyography signals, retraining
Project Title: Electromyography(EMG) signals controlled Assistive Orthotic Robotic Arm for Forearm Movement
Author List:
Mangukiya, Yamik; Graduate, Electrical Engineering, California State University, Fullerton, Presenting Author
George, Kiran; Computer Engineering, California State University, Fullerton
Abstract: There are nearly 1 in 50 people that is approximately six million people affected by paralysis, which restrains the free movements of human body parts. Such people rely on support system that can aid motion and force enhancement to body parts affected by paralysis. The most commonly used support system are prosthetic and orthotic devices. Prosthetics are artificial extensions used to replace the missing body parts or supplement the defective body parts. Whereas, orthotic devices are external mechanical structures that support non-functional body parts which are actuated by motors. This study presents an orthotic robotic arm that can help in retraining flexion and extension movements of forearm of person affected by stroke. For this purpose, surface electrodes are used to acquire the electromyography(emg) signals from biceps brachii of person’s healthy arm. This signals are processed to remove noise and other unwanted signal artifacts and to extract certain statistical features reflecting the forearm movements. Further this signals are used to recognize the forearm movement and identify the states of forearm as a flexion or extension. Based on this states, the actuators of the orthotic robotic arm are controlled. The actuators and supporting robotic arm helps to lift up or lift down the forearm of the paralyzed patients. Also the robotic arm has safety controller for involuntary arm movements, where actuators are not activated if semi flexion or extension movements are detected. Accuracy of device was determined through a series of testing trials on 3 subjects who tested the device for 5 times and device registered 80% accuracy on the total number of trials by the subjects. Preliminary results suggest that the orthotic robotic arm has potential to improve the mobility of hand movements of paralyzed person.
Poster #: 44
Campus: CSU Northridge
Poster Category: Bioengineering
Keywords: lipid droplet, cyanobacteria, carotenoid metabolism
Project Title: Proteomic and transcriptomic support for involvement of cyanobacterial lipid droplets in carotenoid metabolism and sequestration of toxic metabolites
Author List:
Fuentes, Nicole; Graduate, Environmental and Occupational Health, California State University, Northridge, Presenting Author
Arias, Daisy; Graduate, Biology, California State University, Northridge, Presenting Author
Philmus, Benjamin; Oregon State University
Summers, Michael; Biology, California State University, Northridge
Abstract: Lipid droplets (LDs) from the cyanobacterium Nostoc punctiforme are enriched in compounds with biotechnological potential such as neutral lipids, vitamin E, carotenoids, small amounts of a C17 alkanes. To understand the formation and function of these LD associated compounds, isolated LD proteins were subjected to proteomic analysis. This analysis identified Npun_F3745, annotated as phytoene dehydrogenase, an enzyme catalyzing the first rate-limiting step in carotenoid biosynthesis from the colorless isoprenoid compound phytoene. The KEGG database however does not list this protein as part of the carotenoid pathway, so its involvement was investigated. An Npun_F3745 overexpression strain exhibited an abnormally high number of LDs during the exponential phase of growth and isolated LDs contained a large amount of a colorless UV-absorbing compound thought to be an isoprenoid, and low amounts of carotenoids. However, a pigment wavelength scan of the isolated phycobilisome layer of Npun_F3745 over-expressor during LD extraction showed an increase in the presence of carotenoids with a decrease in Phycoerythrin and Phycocyanin compared to the wild-type strain. These results support the hypothesis that Npun_F3745 is involved in carotenoid biosynthesis. A CFP-fusion strain made LDs lacking their normal yellow color, and lack of carotenoids was confirmed by HPLC analysis, thus supporting the hypothesis that the attached CFP blocked multimer formation required for activity. Previous work established that over-production of C17 alkanes resulted in increased LD production, a phenotype similar to the Npun_F3745 overexpression strain. Comparative transcriptome analysis of the alkane over-producer found significant up-regulation of Npun_F3745 that paralleled alkane and LD production, indicating transcriptional regulation of this gene may be used to balance enzyme production with LD over-production. Together these results support: 1) feedback inhibition of enzymes leading to carotenoid synthesis by an unidentified UV-absorbing compound; 2) transcriptional regulation of carotenoid biosynthesis in LDs, and 3) a potential role for LDs in sequestration of toxic hydrophobic compounds. This work has biotechnological applications for future use of cyanobacteria as expression platforms, utilizing LDs to reduce toxicity and allow increased accumulation of useful bioproducts.
Poster #: 45
Campus: CSU Fullerton
Poster Category: Bioengineering
Keywords: multidisciplinary projects, protein kinematics, biomechanics
Project Title: Enhancing Mechanism Kinematics Course through Biomechanics and Biochemistry Experiences
Author List:
Mocanu, Iulian; Undergraduate, Mechanical Engineering, California State University, Fullerton, Presenting Author
Ahir, Vishalkumar; Graduate, Mechanical Engineering, California State University, Fullerton, Presenting Author
Robson, Nina; Mechanical Engineering, California State University, Fullerton
Abstract: This project discusses results of introducing faculty research in the areas of human and protein kinematics within a junior engineering Kinematics of Mechanisms course, with the main goal of preparing the students to be critical thinkers, multidisciplinary problem solvers and life-long learners. Traditionally, the Kinematics of Mechanisms is taught as a focused lecture course for mechanical engineers, presenting knowledge on analysis, design and construction of mechanical systems, without any reference to kinematics of existing mechanisms in Biotechnology in general. During the past year, new interactive activities and multidisciplinary research projects were developed and incorporated within the course. These activities were specifically designed to enhance the students’ knowledge of how the above-mentioned mechanical systems appear in other biotechnological domains as models of locomotive organisms, human limbs or molecular structures, among others.
Student performance as well as the three desired learning outcomes, critical thinking, intellectual maturity, and responsibility for own learning, were assessed through anonymous surveys based on students’ and faculty perception. Within the surveys, students were required to outline questions that they were asking themselves while working on each project. The questions were then categorized according to relevance to the desired outcomes.
A comparison between the results from two semesters, before and after the incorporation of the new activities, showed that presenting a series of multidisciplinary projects, designed specifically to complement each other, improved the students’ intellectual growth by 5% and responsibility for their own learning by 3%. In addition, the students’ performance on the project content and presentation increased with more than 10% as assessed from the average student grades on projects and course grades. These results show that the developed methods prove efficient not only for learning new material, but also in transferring earned skills to tasks of greater difficulty, i.e. tasks in different domains, such as Biotechnology.
The idea of enhancing a junior mechanical engineering course with research activities related to application of knowledge to different bio-related domains is novel. In future, with sufficient changes the new activities could be adopted in different biotechnological courses.
Funding: Health Promotion Research Institute, CSUF
Poster #: 46
Campus: San José State University
Poster Category: Bioengineering
Keywords: Platelets, Storage, Clot biomechanics
Project Title: Platelet Storage: A Chilling Story of Structure and Function
Author List:
Ramasubramanian, Anand; Biomedical, Chemical and Materials Engineering, San José State University
Nair, Prajeeda; University of Texas at San Antonio
Dastghaeib, Amir; Undergraduate, Biomedical, Chemical and Materials Engineering, San José State University, Presenting Author
Schobey, Stephen; Undergraduate, Biomedical, Chemical and Materials Engineeringe, San José State University, Presenting Author
Cap, Andrew; US Institute of Surgical Research, San Antonio, TX
Abstract: BACKGROUND: Functional platelets are critical in hemostasis. At the site of injury, activated platelets generate thrombin that converts soluble plasma fibrinogen to polymeric fibrin clot; and then actively contract the clot to increase the strength so as to effectively dam excessive bleeding. Currently, platelets are stored up to 5 days at room temperature (RT) with gentle agitation. However, this standard of care is suboptimal due to progressive loss of platelet function, increased risk of bacterial contamination, poor shelf-life, and transportation logistics. Cold storage (4 °C) of platelets may mitigate these issues, and holds promise as a superior transfusion product. We hypothesized that 4 °C-stored platelets will generate clots with superior mechanical properties compared to those from RT-stored platelets.
METHODS: Apheresis platelets were collected healthy donors and stored for 5 days at RT or 4°C. The rheological properties including clot stiffness, strength, and contractile forces were estimated by cone-and-plate rheometry. The structural properties of the clots developed from fresh platelets or stored platelets were analyzed by scanning electron microscopy, and various attributes such as fibrin diameter, density and tortuousity or curvature were quantified. Platelet-fibrinogen interactions were evaluated by fluorescence microscopy, and thrombin generation was assayed. The experiments were repeated with at least three donors in triplicates, and one-way repeated measures ANOVA with post hoc Tukey’s or a paired t-test. The groups were considered significant for p- values<0.05.
RESULTS: We observed that clots formed from (i) cold-stored platelets have clot stiffness and strength similar to that fresh of platelets, and 2-3 fold higher than those from RT-stored platelets; (ii) cold-stored platelets were characterized by thinner, shorter and less tortuous fibers and increased fibrin density compared to RT-stored platelets; (iii) cold-stored, but not RT-stored, platelets adhered and spread on fibrinogen-coated surfaces; and (iv) stored platelets generated more thrombin than fresh platelets.
CONCLUSION: Cold stored platelets form clots with superior rheological properties compared to RT-stored platelets, which is a result of the underlying fibrin architecture due to better platelet-fibrinogen interactions and thrombin generation. Thus, cold stored platelets may be a viable storage alternative associated with better outcome post trauma.
Poster #: 47
Campus: CSU Long Beach
Poster Category: Bioengineering
Keywords: microfluidics, 3D printing, microbeads
Project Title: Alginate Microbead Synthesis on an 3D-printed, Automated Platform
Author List:
Esqueda, Genesis; Undergraduate, Chemical Engineering, California State University, Long Beach, Presenting Author
Grosvirt-Dramen, Adam ; Undergraduate, Chemical Engineering, California State University, Long Beach, Presenting Author
Venkatraman, Rahul; Graduate, Chemical Engineering, California State University, Long Beach
Lo, Roger; Chemical Engineering, California State University, Long Beach
Abstract: The role of microbeads in biomedical applications is continuing to increase due to their relatively large surface area and adaptability for drug delivery vehicles, cell encapsulation, single-cell studies, and medical diagnostics and imaging. The biocompatibility of alginate makes it a viable material for microbeads used in these applications. However, traditional methods for the microbead synthesis have shown to be time-consuming and ineffective for continuous synthesis. This study aims to develop an automated platform for continuous microbead synthesis by means of 3D printing and microfluidics. The 3D-printing technology is used to reduce the manufacturing time and costs of the microfluidic devices for high-throughput production of alginate microbeads with precise control over size and shape. Alginate microbeads were generated on a 3D-printed microfluidic device by emulsifying an aqueous alginate solution in an oil phase, followed by ionic crosslinking. We have designed and fabricated the first prototype microfluidic device and successfully carried out a continuous synthesis that can produce microbeads of 577.19 +/- 5.46 microns in diameter. A new design is being developed for smaller beads. This microbead synthesis is now controlled by manually adjusting the flow rates of reagents from the pump control panel. We are currently developing a control unit using the open-source Arduino board and Python language to automate the synthesis process.
Poster #: 48
Campus: CSU Fullerton
Poster Category: Bioengineering
Keywords: Quality-of-life, Navigation, wheelchair
Project Title: Autonomous Wheelchair Navigation System for Individuals with Severe Disabilities
Author List:
Grewal, Harkishan Singh; Undergraduate, Computer Engineering, California State University, Fullerton, Presenting Author
Matthews, Aaron Preston; Undergraduate, Computer Engineering, California State University, Fullerton
Tea, Richard Jayavarman; Undergraduate, Computer Engineering, California State University, Fullerton, Presenting Author
George, Kiran; Computer Engineering, California State University, Fullerton
Abstract: Electric wheelchairs restore mobility and independence to those with injuries limiting their movements. However, people suffering from motor neuron degeneration diseases, such as Amyotrophic lateral sclerosis (ALS), lack the fine motor control to benefit for electric wheelchairs, leaving them dependent on others and reducing their quality of life. In this study, we seek to mitigate this problem by developing a wheelchair capable of autonomous navigation, with minimal input form the user. We use robot operating system (ROS), micro-controllers, rotary encoders, and a LIDAR unit to modify an electric wheelchair. The LIDAR unit provides measurements of the environment which are used to develop a map of the surroundings. The micro-controllers take input from the LIDAR and rotary encoders to develop a navigation path to a target location. The micro-controllers send signals to the control system of the wheelchair to move it along the designed path. We configured the ROS software to adapt to changes in the environment and reroute the wheelchair if new obstacles emerge. We tested the navigation system in three different environments: straight path with no obstacles, zigzag path with static obstacles, and zigzag path with static and dynamic obstacles. We conducted five trials for each environment. The wheelchair navigated the straight path with no obstacles 100% of the trials, with an average time of 30 seconds and path length of 11.73 meters. The wheelchair navigated the zigzag path with static obstacles 80% of the trials, with an average time of 30.6 seconds and path length of 12.13 meters. In the zigzag path with dynamic and static obstacles, the wheelchair successfully navigated 80% of the time with an average time of 42.5 seconds and path length of 15.71 meters. The wheelchair suffered a localization error during trial 1 and 5, where it was unable to localize its initial position. The wheelchair recovered and completed trial 1, but was unable to do so in trial 5. Based on these preliminary results, the autonomous navigation system shows potential to navigate a variety of environments with hope of restoring complete indoor mobility. We hope to correct for the localization error and add more sensors to give the wheelchair a refined sense of the environment. We also hope to test the navigation system in more varied environments with wider range of obstacles.
Poster #: 49
Campus: CSU Long Beach
Poster Category: Clinical
Keywords: Addiction, animal model, behavior
Project Title: Alcohol reward in periadolescent male and female rats
Author List:
Bates, Emily; Graduate, Psychology, California State University, Long Beach, Presenting Author
Yang, Yeji; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Pentkowski, Nathan; University of New Mexico
Zavala, Arturo; Psychology, California State University, Long Beach
Abstract: Alcohol is the most commonly abused recreational substance that is first consumed during adolescence. Preclinical research examining the rewarding effects of alcohol in adolescence, as assessed using the conditioned place preference (CPP) paradigm, have resulted in weak or inconsistent results. The present study sought to examine whether ascending doses of alcohol during conditioning produces more robust alcohol-induced CPP compared to a fixed dosing regimen during conditioning. Support for this hypothesis is indicated by recent studies demonstrating more robust cocaine-induced CPP using ascending doses of cocaine during conditioning compared to fixed doses during conditioning. Beginning on postnatal day (PD) 23, male and female periadolescent rats underwent a 10-day alcohol CPP procedure. On days 1 and 10, rats were tested for their pre-conditioning and post-conditioning place preferences, respectively, during 15-min sessions. On days 2-9, rats were conditioned for 15-min with saline or alcohol on alternative days. During alcohol conditioning days, rats were randomly assigned to receive either ascending alcohol doses (0.125-2.0 mg/kg, intraperitoneally) or fixed alcohol doses (0.5, 1.0, or 2.0 mg/kg, intraperitoneally). Results reveal differences in the strength of CPP in a dose dependent manner between fixed vs ascending dosing of alcohol during conditioning, with the latter producing more robust CPP. Overall, these results suggest that the pattern of doses used during conditioning sessions may be play an important role in elucidating the rewarding effects of alcohol.
Poster #: 50
Campus: CSU Monterey Bay
Poster Category: Clinical
Keywords: T-cell, Immunotherapy, Galectin
Project Title: Galectin-3 Inhibitor GR-MD-02 Increases CD8 T Cell Infiltration of MCA-205 Tumors
Author List:
Martin, Leah; Undergraduate, Biology, California State University, Monterey Bay, Presenting Author
McNamara, Michael; Postdoc, Robert W. Franz Cancer Research Center
Linch, Stephanie; Postdoc, Robert W. Franz Cancer Research Center
Kasiewicz, Melissa; Staff, Robert W. Franz Cancer Research Center
Hilgart-Martiszus, Ian; Staff, Robert W. Franz Cancer Research Center
Farhad, Mohammad; , Robert W. Franz Cancer Research Center
Traber, Peter; Galectin Therapeutics Inc.
Redmond, William; Robert W. Franz Cancer Research Center
Abstract: Galectin-3 (Gal3, lgals3) is a carbohydrate-binding protein that is produced by both leukocytes and cancer cells. Serum Gal3 levels have been shown to be elevated in patients with metastatic melanoma relative to patients with non-metastatic melanoma. These increased Gal3 levels are thought to help promote metastasis and angiogenesis of tumors. Gal3 has also been found to inhibit tumor-infiltrating lymphocytes (TIL) by disrupting T-cell receptor (TCR) signaling, while promoting anti-inflammatory functions (M2 polarization) in macrophages. Gal-3 has been associated with various pathological and physiological processes that play a role in cell differentiation and growth, cell adhesion, chemo-attraction, apoptosis and cell cycle regulation. The purpose of this research project is to better understand how a Gal3 inhibitor, GR-MD-02, impacts the tumor microenvironment and whether GR-MD-02 is synergistic with an OX40 agonist mAb (OX86). In this experiment, tumor-bearing mice were treated with an IgG control, OX86, GR-MD-02 or OX86 + GR-MD-02. Tumors were harvested from these mice and analyzed by Immunohistochemistry (IHC) and microarray transcriptome profiling. These experiments resulted in three key findings:
1) GR-MD-02 was present in the tumor, but did not appear to be associated with T Cells.
2) Both GR-MD-02 and OX86 increased CD8 T cell infiltration into the tumor, but had minimal impact on the infiltration of CD4 T cells or Macrophages.
3) Expression of several immune checkpoint receptors were up-regulated in the tumor following treatment.
Poster #: 51
Campus: Cal Poly San Luis Obispo
Poster Category: Clinical
Keywords: Type VI secretion system, Dienes line, Vibrio parahaemolyticus
Project Title: Type VI Secretion System Expression of Vibrio parahaemolyticus in Intraspecific Competition
Author List:
Campbell, Alexandra; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Leak, Megan; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Yeung, Marie; Biological Sciences, California Polytechnic State University, San Luis Obispo
Abstract: The rise of antibiotic-resistant bacteria has reached an alarming level. New ideas must be explored to address this concern. Recently, a new secretion system called type VI secretion system (T6SS) was found in different organisms that appears to be a defense mechanism. Previous studies suggest Vibrio spp. may express T6SS. In this study, Vibrio parahaemolyticus, a common foodborne pathogen in US, is used as a model to study T6SS. The objectives of this study were to evaluate culturing conditions that would stimulate swarming motility in strains of V. parahaemolyticus and to detect for the presence of T6SS based on phenotypic criteria. Different media (brain heart infusion, tryptic soy agar with salt), varying agar concentration (0.7%, 1%, 2%) and incubation temperature (25 and 37 degrees Celsius) were tested to determine the optimal environment that induces swarming in 15 V. parahaemolyticus strains. The diameter (in cm) of growth from the center on a 150-mm agar surface was measured to quantify swarming in varying conditions. The greatest extent of swarming was observed when V. parahaemolyticus strains were incubated in BHI medium containing 1% agar at 25 degrees Celsius. In these conditions, the strains swarmed on the entire agar surface within 3 days. When two T6SS-positive strains of the same species swarm towards each other, instead of forming a continuous mass, a Dienes line forms at the junction indicating competition between the two strains. Thus, the next step was to detect Dienes line formation. Pairwise testing was performed from the 15 strains, with each pair inoculated 0.5 cm apart on the agar surface. Pairwise testing of identical strains served as the negative control while Proteus mirabilis was used as the positive control. Eight strains (53%) of V. parahaemolyticus were positive for Dienes line formation under the test conditions. Primers for T6SS genes were designed, which will be used to investigate the expression of T6SS in these Dienes line-positive strains using RT-qPCR. Understanding the conditions of when and how T6SS is expressed will help us develop strategies to harness this secretion system to target antibiotic-resistant pathogens. Funding provided by the Baker and Koob Endowments of Cal Poly SLO.
Poster #: 52
Campus: Sacramento State University
Poster Category: Clinical
Keywords: Retinoid, Sebum, Gas Chromatography
Project Title: Retinoid-Induced Changes in the Fatty Acid Profile of Sebum and Its Effect on Bacterial Growth
Author List:
Andersen, Erich; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Valdez, Nico; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Crawford, Robert; Biological Sciences, California State University, Sacramento
Abstract: Dysbiosis of the skin microbiome contributes to the pathogenesis of acne vulgaris. Clinical administration of retinoids have been demonstrated as an effective therapeutic but the role of these compounds in altering the local lipid profile and the growth of local bacteria are not well understood. We hypothesize here crosstalk between treatment-induced lipid secretion and bacteria found in acne patients. Human subjects with acne were recruited in two groups where one group received topical tretinoin and the other group received oral isotretinoin. Facial sebum was collected through the use of Sebutapes at baseline and after 1 month of therapy, and sebum excretion was measured with a Sebumeter. Sebum was extracted and fatty acid profiles were quantified by gas chromatography with a flame ionization detector. Clinical isolate strains Staphylococcus epidermidis and Staphylococcus aureus were analyzed hourly for growth over a 12 hour span in the presence or absence of prominent fatty acid fractions determined by GC-FID. Our results suggest that tretinoin did not alter overall sebum production but isotretinoin reduced sebum production by 7.1-fold (p < 0.01). The relative free fatty acid fractions for C12:0 and C16:0 were unchanged; C14:0 was decreased [1.2-fold, p < 0.01]; and C18:0 was increased [1.5-fold, p < 0.01] after one month of topical tretinoin therapy in comparison to baseline. The relative free fatty acid fractions for C12:0 and C16:0 were unchanged; C14:0 was decreased [1.7-fold, p < 0.01]; and C18:0 was increased [2.3-fold, p < 0.01] after one month of oral isotretinoin therapy in comparison to baseline. Growth of S. epidermidis was reduced 10-fold [p < 0.01] by C14 and 50-fold by C18 [p < 0.01], respectively. However, the growth of S. aureus with C14 and C18 was more profoundly impaired relative to S. epidermidis grown in the same free fatty acid fractions [100-fold, p < 0.01]. Interestingly, transcriptomic analysis using Next Generation Sequencing Technologies showed lipid and species-specific gene expression changes. Collectively, these results indicate that topical and systemic retinoids alter free fatty acid profiles of sebum independent of their effect on the overall sebum production rate and induce changes to growth and gene expression of local bacteria. These studies were funded by a UC Davis-CSUS Seed Grant.
Poster #: 53
Campus: CSU Long Beach
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Biomedical Signal Processing, Pulse Latency Onset Detection, Cerebral Blood Flow
Project Title: Cerebral Blood Flow Velocity Pulse Onset Latency Estimation: A Comparative Study
Author List:
Arevalo, Natalie; Undergraduate, Biomedical Engineering, California State University, Long Beach, Presenting Author
Scalzo, Fabien; Department of Neurology, University of California, Los Angeles
Hamilton, Robert; Neural Analytics, Inc., Los Angeles
Hanchey, Dan; Neural Analytics, Inc., Los Angeles
Asgari, Shadnaz ; Biomedical Engineering, California State University, Long Beach
Abstract: Intracranial Pressure (ICP) is the pressure inside the skull, the brain, and cerebrospinal fluid. If ICP level surpasses a normal range of 7-15 mmHg, brain tissue could be irreversibly damaged leading to brain death. Currently, ICP is measured through an invasive procedure which carries various risks and complications including bacterial infections and further hemorrhaging. Cerebral Blood Flow Velocity (CBFV) signal captured noninvasively through transcranial Doppler, has been shown to carry predictive information about the ICP level. Recent studies indicate that CBFV pulse onset latency can be used as a feature to estimate ICP noninvasively. However, CBFV signal is inherently contaminated with noise and artifacts that obscure the accurate estimation of its pulse onset latency. In this project, we aimed to identify a robust method to accurately estimate the pulse onset latency of CBFV signal. For this purpose, we implemented two different methods, the Farooq method and the Chen method, that were originally developed for the pulse onset latency estimation of signals other than CBFV, e.g. photoplethysmographic signals. We manually annotated the pulse onset latencies of a large dataset of 91,465 CBFV pulses using a software developed in-house. The CBFV data was collected from 111 patients who were admitted to UCLA Medical Center for subarachnoid hemorrhage evaluation. We applied each method to our dataset and estimated the CBFV pulse onset latencies of all the pulses. To evaluate the accuracy of each method, the sensitivity and positive predictivity value (PPV) were calculated by comparing the estimated results with those of the manual annotation. The results showed that the Farooq method has the higher sensitivity of 94.99% (versus 85.9% for the Chen method). The Farooq method also achieved the higher PPV of 98.8% (versus 97.1% for the Chen method). However, the running time of the Chen method was fifteen times lower than that of the Farooq method (31 seconds versus 442 seconds). We conclude that although the Farooq method achieves an acceptable level of accuracy for pulse onset latency estimation, a further improvement of its algorithm in terms of computational complexity may be needed to make it more suitable for real-time application.
Acknowledgement: This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers; 8UL1GM118979-02; 8TL4GM118980-02; 8RL5GM118978-02.
Poster #: 54
Campus: Sacramento State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: enediyne cyclization, density functional theory (DFT), anticancer
Project Title: Steric Effects in the Computational Modeling of Cyclization Reactions of Enediynones
Author List:
Hoang, Kim Anh; Undergraduate, Chemistry, California State University, Sacramento, Presenting Author
Li, Cui; Graduate, Chemistry, California State University, Sacramento
Gherman, Benjamin; Chemistry, California State University, Sacramento
Spence, John; Chemistry, California State University, Sacramento
Abstract: The term enediyne refers to organic compounds containing a carbon-carbon double bond lying between two carbon-carbon triple bonds. These compounds can undergo Bergman cyclization to form transient benzenoid diradicals that have the ability to abstract hydrogen atoms from the deoxyribose moiety on the backbone of DNA strands, causing DNA strand cleavage and inducing cell apoptosis. As a result, enediynes have been proposed as a core for anticancer drugs. To increase the range of cyclization reactivity, the enediyne is modified by insertion of a carbonyl group, producing an enediynone. This new compound can undergo cyclization along four different pathways, generating new diradical intermediates with simultaneous formation of a 5-, 6-, or 7-membered ring. Previous experimental study on the cyclization reactivity of unsubstituted enediynones yielded a complicated mixture of products. Therefore, the aim of this project is to make the product mixture more readily analyzable by adding substituents to provide steric bulk at the alkyne termini. This is intended to disfavor cyclization pathways involving those positions. Density functional theory (DFT) calculations are used to determine the energetics of the four reaction pathways of phenyl and naphthyl monosubstituted and disubstituted enediynones to determine the preferred reaction pathways in each case. To account for dispersion interactions between substituents which occur upon cyclization, empirical dispersion corrections are included within the DFT calculations. A correction based upon coupled-cluster calculations accounts for DFT errors in the interaction between the diradicals and any aromaticity that develops in the newly formed rings. Data show that the addition of aryl substituents to the alkyne termini raises the reaction energetics for pathways involving those positions, while lowering the energies of pathways involving the unsubstituted positions. In addition, the effect of substituents is on average greater for the reaction energies than for the activation energies. Further, both the dispersion and coupled-cluster corrections are shown to appreciably affect the original DFT reaction energetics and thus to be important aspects of obtaining accurate calculations for enediynone cyclization reactions.
Acknowledgements: Funding for this project has been provided by California State University, Sacramento to K. A. H. through the McNair Scholars Program and a Russell-Forkey Research Award.
Poster #: 55
Campus: CSU Fullerton
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: dry-eye diseases, fast clinical detection, tear film
Project Title: Mathematical Model to Noninvasively Detect Dry-Eye Diseases
Author List:
Nguyen, Trini; Undergraduate, Mathematics, California State University, Fullerton, Presenting Author
Lee, Charles; Mathematics, California State University, Fullerton, Presenting Author
Abstract: Wavelength-dependent interferometry is a non-invasive technique that can accurately diagnose patients with dry-eye diseases. The method requires shining a light onto the surface of the eye while measuring its reflectance, which then allows for the thicknesses of the tear film layers to be extracted implicitly based on the resulting reflectance. Since there are several layers of the tear film overlaying the cornea, light must be emitted at multiple wavelengths to produce sufficient degrees of freedom to determine the thicknesses of the lipid and the aqueous layers of the eye. In a 2013 patent, a mathematical model for the measurement of the reflectance is presented. However, the model only fitted well in some cases and not for other sets of data because it lacked the usage of the scalar light scattering theory, which accounts for the light that is scattered at an optically rough surface. Instead, the author of the patent added an ad-hoc exponential term to the model to mimic the light scattering effect in order to force the model to fit well with the collected data. As a result, the model is empirical rather than physical. In this work, a mathematical model that describes the reflectance of the eye is derived. The model takes into account electromagnetic properties of the light that is transmitted through several different layers and reflected back to the top layer. Specifically, the electromagnetic fields at the interfaces must match up at each layer under the Snell’s laws depending on material properties, thicknesses, and the incident, reflectance and transmittance angles. In addition, a numerical scheme based on this preliminary mathematical model has been implemented, which was quite complicated in the patent. The provided algorithm in the patent takes up over ten pages of code and over 45 minutes in time to produce the thicknesses. Our scheme can produce the thicknesses of the different layers of the tear film of the eye within a few seconds, rendering its applicability for a swift and simple clinical diagnosis of dry-eye diseases. Our future work will include refining the application of the scattering theory into the mathematical model as well as improving the speed of the algorithm.
Funding: MARC, NIH.
Poster #: 56
Campus: CSU Fresno
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Mathematical Biology, Insect, Simulation
Project Title: Field imaging reveals perceptual and navigational strategies of wood ants in naturalistic environments
Author List:
Arevalo, Erik; Graduate, Biology, California State University, Fresno, Presenting Author
Warnert, Reina; Undergraduate, Biology, California State University, Fresno, Presenting Author
Mendoza, Austin; Undergraduate, Biology, California State University, Fresno
Lent, David; Biology, California State University, Fresno
Abstract: When an animal moves through their environment they must identify reliable sensory cues to guide their routes. Identifying reliable sensory cues is not a trivial process because often it is not possible to know in advance which cues may be useful. Animals that rely on vision to navigate through the world must extract and integrate behaviorally relevant visual cues, often from cluttered scenes, so that they can locate a desired goal. In ants, a number of studies have identified several visual cues that can guide an ant when navigating, however, as to how the cues are perceived or prioritized is unknown. Our study aims to understand how ants value cues and how specific cues or combination of cues comes to be learned. In order to investigate visual processes in ants, we used image processing and computational studies of natural scenes. First we simulated a foraging route in MATLAB using a Levy walk, which is a mathematically optimized search strategy, and generated series of X and Y points to be mapped out in the field. With these points determined, we went in to the field to collect images for analysis. We took a series of panoramic images that were 360 by 80 degrees along a simulated foraging route in a wooded area (Allergy Free Garden) on the Fresno State campus. Once images were collected we processed them through our algorithms that: convert to ant visual resolution of approximately 4 degree and extract features that have been experimentally shown to guide an ants route such as edges, peaks, centroids. Once image stacks from the route were collected and processed, we searched for the cues that were most stable during the random walk. Our study of navigation over both short distances and long distances suggest that the edge information was sufficient to guide the ant on subsequent routes. That is, if the ants only used a stored representation of the edges during subsequent foraging events they were able to get from start to end in a relatively straight trajectory using image matching. These results have provided insight into the mechanisms involved in prioritization and perception of visual information. This study also demonstrates that image-matching foraging can be accomplished with storage of minimal information. Several replications of new foraging routes are being completed to support data. Additionally, this work has let us investigate how memory can be optimized in simple networks and nervous systems
Poster #: 57
Campus: San Diego State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Molecular Dynamics, Protein Design, Thermal Stability
Project Title: Computational Study of the Effects of Variable Temperatures on Protein Secondary Structure and Stability
Author List:
Chittoor Prem, Aishani; Graduate, Biomedical Informatics, San Diego State University, Presenting Author
Suon, Peter; Graduate, Chemistry and Biochemistry, San Diego State University
Lokensgard, Melissa; Graduate, Chemistry and Biochemistry, San Diego State University
Love, John; Chemistry and Biochemistry, San Diego State University
Abstract: Comparing the folding mechanisms of proteins with similar secondary structures yet with different sequences can provide fundamental insights in understanding important properties of proteins. Molecular Dynamics (MD) is a computational tool used to analyze protein dynamics and, in our study, the thermal stability of proteins on an atomistic level. Here, we describe the use of the AMBER 14 MD package to carry out computer simulations on two sets of protein mutants in order to understand the effect that point mutations (in the core of a protein) have on surrounding secondary structure elements and on overall protein thermal stability. The two sets of proteins are mutants of the β1 domain of streptococcal protein-G and the human protein ubiquitin. MD simulations were conducted on a series of different mutants at simulated temperatures that ranged from 200K to 500K in 25K increments. The purpose was to study and analyze the energies and conformational changes that occur within the series of mutant test proteins at increasing temperatures. The results of these calculations significantly enhanced our understanding of the effect that core point mutations have on β-hairpin secondary structure elements common to both test proteins. The resulting MD trajectories were analyzed for retention of main-chain hydrogen bonds for the β-hairpin secondary structure elements as a function of increasing temperature. In a number of interesting cases, point mutations in the hydrophobic core reduced the residence time of certain main-chain hydrogen bonds for positions that were not in close proximity to the mutations. Computational evidence complimenting experimental results entailing the thermal stability of these mutants was found by analyzing their conformational changes during the course of these simulations. A major focus of this project is to adapt this form of dynamic simulations as a useful approach for providing computational results that are complementary to experimental data. The goal is to apply rigorous analysis that can be used as a pipeline for guiding protein design projects by providing a robust bridge between theory and experiment.
Poster #: 58
Campus: CSU Monterey Bay
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Cancer, Machine Learning, RNA-seq
Project Title: A Comparison of Different Machine Learning Algorithms for Predicting Gene Essentiality in Cancer Cell Lines
Author List:
Hansen, Blake; Undergraduate, Math and Statistics, California State University, Monterey Bay, Presenting Author
Jue, Nathaniel; School of Natural Sciences, California State University, Monterey Bay
Abstract: Cancer is a heterogenous disease, arising from many different genetic mutations, producing many different unique types of tumors, each dependent upon different genes to maintain its cancerous phenotype. Reliable methods of predicting gene essentiality could identify these unique weaknesses, allowing doctors to design therapies that specifically targets these weaknesses with minimal harm to the patient’s healthy cells. The purpose of this study is to determine which machine learning algorithm or combination of algorithms can most effectively predict gene essentiality from genomic data. We expect that an ensemble of different approaches will predict gene essentiality more accurately than any individual approach. Cancer Cell line data was collected from the Cancer Cell Line Encyclopedia (gene expression and copy number variation) and the Achilles Project (shRNA-based gene essentiality measurements). Three different algorithms (Random Forest (RF), Multiple Pathway Learning (MPL), glmnet) were trained on a subset of the data and used to predict essentiality in the withheld samples. Two of the methods (RF and glmnet) directly use genomic data as its features, while MPL first aggregates genomic data on the pathway level and then uses the pathway aggregates as features. Predictions for each gene across withheld samples were correlated with the gene essentiality scores measured by the Achilles Project via spearman correlation. Models were compared based on their mean correlation across all genes. The model that had the highest mean spearman correlation with the Achilles Project measurements was 0.55 RF, 0.40 MPL, and 0.05 glmnet with a mean correlation of 0.30, with correlations for individual genes as high as 0.70.
Poster #: 59
Campus: CSU Long Beach
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: bioinformatics, fungi, biofuel
Project Title: Enzymes for cellulose, xylan, and chitin deconstruction in fungi
Author List:
Berlemont, Renaud; Biological Sciences, California State University, Long Beach, Presenting Author
Abstract: In many environments, fungi are important for the degradation of polysaccharides. This process is central to nutrients mobilization and carbon cycling. The breakdown of polysaccharides by fungi requires the production of glycoside hydrolases (GH) and oxidative enzymes (LPMO), sometime associated with accessory non-catalytic domain (e.g., CBM). Here the taxonomic distribution and the diversity of 10,254 proteins targeting cellulose, xylan, and chitin from 203 sequenced fungal genomes are described. The potentials to degrade cellulose, xylan and chitin, based on annotated genes, are conserved within genera. However, even between closely related taxa (e.g., species) many identified enzymes display unique domain architecture. Among others, we identified multi activity proteins with multiple catalytic domains and endowed with the potential to target multiple substrates (e.g., cellulase-xylanase). These enzymes with unique domain association are interesting potential new catalysts for industrial polysaccharide deconstruction (e.g., biofuel industries).
Poster #: 60
Campus: CSU San Marcos
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Population genetics, Biocontrol, Bioinformatics
Project Title: Demographic histories of three predatory lady beetles reveal complex patterns of diversity and population size change in the United States
Author List:
Sethuraman, Arun; Biological Sciences, California State University San Marcos
Vasquez, Yumary; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Rubio, Michael; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Janzen, Fredric; Iowa State University
Obrycki, John; University of Kentucky
Abstract: Predatory lady beetles (Coccinellidae) contribute to biological control of agricultural pests, however, multiple species frequently compete for similar resources in the same environment. Numerous studies have examined the ecological interactions of the native North American convergent lady beetle (Hippodamia convergens) and two introduced species, the seven spotted lady beetle (Coccinella septempunctata) and the Asian lady beetle (Harmonia axyridis), in agricultural fields and described multi-year population dynamics. However, the evolutionary dynamics of these interacting predatory beetles are uncharacterized. Here we utilize publicly available multi-locus genotype data (mitochondrial and microsatellite) from geographically disjunct populations of these three species to estimate demography across populations in North America. Coalescent analyses reveal (1) a recent (~4-5 years) decline (>12 fold) in population size of H. convergens, (2) a massive (>150 fold), and very recent, population size decline in Ha. axyridis, and (3) population size growth (mutation scaled growth rate in 1/u generations = 997 SD = 60)) over recent and evolutionary time scales in C. septempunctata. Considering that these estimates are based on two different types of genetic data with differences in mutation rates (mitochondrial versus nuclear), these dynamic and differing population size histories warrant further studies of the interactions of these predatory lady beetles in the field, and consequences of population size change for evolutionary trajectories in North America.
Poster #: 61
Campus: Cal Poly San Luis Obispo
Poster Category: Diagnostics/Imaging/Analytical
Keywords: skin tissue, reactive oxygen species, immunofluorescence
Project Title: Sphingomyelin Protects Keratinocytes from UVB Induced Reactive Oxygen Species
Author List:
Kandell, Rebecca; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Kandell, Jennifer; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Shah, Nikita; Graduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Laiho, Lily; Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Abstract: Skin, the largest organ of our bodies, prevents environmental pathogens and toxic agents from entering. Skin also protects the human body from ultraviolet (UV) radiation, which significantly contributes to skin cancer. The two most frequently occurring types of skin cancer are basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). The latter originates from keratinocytes and is more likely to spread to other organs in the body. Currently, the best chemical filter that protects skin from absorbing UV radiation is sunscreen. Unfortunately, by-products from degradation of sunscreen can be carcinogenic themselves. Organic alternatives to chemical filters are needed to replace chemical UV blockers. Sphingomyelin (SM), a phospholipid in cell membrane and signaling molecule, may be used to protect keratinocytes. After a 24-hour incubation with SM, keratinocytes were exposed to UV radiation, which induces reactive oxygen species (ROS). ROS harms macromolecules and DNA within the cell and can be stained for visualization and imaged. Fluorescence intensities for each treatment group (+UV/-SM; +UV/+SM; -UV/+SM; -UV/-SM) were compared and there is a significant reduction in ROS production when UV exposed keratinocytes are treated with sphingomyelin (+UV/+SM) than without treatment (+UV/-SM). Keratinocytes exposed to UV radiation and treated with sphingomyelin produced less reactive oxygen species than when not treated with sphingomyelin. This implies that sphingomyelin may help to deter the harmful effects of UV radiation.
Poster #: 62
Campus: CSU Los Angeles
Poster Category: Diagnostics/Imaging/Analytical
Keywords: paper microfluidics, acetylcholinesterase, assay
Project Title: Thread-Based Microfluidic Chips as a Platform to Assess Acetylcholinesterase Activity
Author List:
Gaines, Michelle; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Gonzalez, Ariana; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles
Gomez, Frank; Chemistry and Biochemistry, California State University, Los Angeles
Abstract: The quantitative detection of acetylcholinesterase (AChE) by colorimetric analysis utilizing a microfluidic thread-based analytical device (µTAD) is described. It can be difficult to study neurotransmitters (e.g. acetylcholine [ACh]) directly and, in such cases, the activity of AChE can be studied. Levels of ACh are relevant to Alzheimer’s disease as ACh levels can drop by as much as 90% in some patients. One way to compensate for lower levels of ACh is to inhibit the AChE enzyme from breaking down ACh. Hence, the development of facile and convenient methods to both detect AChE and screen potential inhibitors will aid in the diagnosis (and eventual cure) for Alzheimer’s disease. In the present work, two µTAD platforms were fabricated. Both had a nylon thread trifurcated into three channels terminating at open sites at the ends of the thread. One platform had one end of the thread split into two, with the third end cut off. The nylon on the second platform was not trifurcated at the other end, creating a single inlet. 5,5’-Dithiobis-(2-nitrobenzoic acid) (DTNB) was spotted and dried on the analysis sites. Acetylthiocholine iodide (ATC) (or cysteine, Cys), is transported through one channel of the nylon thread by capillary action due to the hydrophilic nature of nylon. AChE is transported through the other channel of nylon thread and mixes with the ATC (or Cys) as they travel up to the analysis sites. As the solution reaches the analysis sites, an intense yellow color change occurs indicating the reaction of the thiol with DTNB to produce the yellow anion TNB2-. The sites are then dried, scanned, yielding a linear range of inverse yellow mean intensity versus substrate concentration. An IC50 value (1.741 nM) with a known inhibitor, neostigmine bromide (NB), is obtained on the device. The multiplex design enables triplicate data collection in a device that is easy to use. µTADs have great potential to be employed in a myriad of tests including point-of-care (POC) diagnostic devices for resource-challenged settings.
Poster #: 63
Campus: CSU Stanislaus
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Corrole, Solvent Effect, Photochemistry & Photophysics
Project Title: Photophysical, Electrochemical, and Spectroelectrochemical Characterization and Solvent Effect on the Tautomerism of Free-Base Corrole
Author List:
Calvillo, Guadalupe; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Reed, Christopher; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Kohl, Forrest; Undergraduate, Chemistry, California State University, Stanislaus
Klein, Silvia; Undergraduate, Chemistry, California State University, Stanislaus
Loogman, Anthony; Undergraduate, Chemistry, California State University, Stanislaus
Lupercio, Stephanie; Undergraduate, Chemistry, California State University, Stanislaus
Alemán, Elvin; Chemistry, California State University, Stanislaus
Abstract: Corroles are aromatic compounds consisting of four pyrrole-like subunits connected in a ring via three methane bridges and one direct bond between the alpha carbons of two of the subunits. They have a structure and photophysical properties similar to that of porphyrins, which are currently being researched in blocking tumor progression and metastasis. In photosynthetic bacteria, tetrapyrrolic molecules are used in the light harvesting complexes to channel solar energy towards the reaction complex where photosynthesis occurs. Similar to porphyrins, corroles demonstrate exceptional photophysical properties in regards to their absorbance of light and fluorescence. Corroles exist in two separate tautomeric states causing them to exhibit noticeably different photophysical and electrochemical behaviors in different solvents. A proper understanding of corroles interaction with solvents and how they affect corroles light absorbing emitting capabilities are necessary to develop potential uses of corrole in artificial photosynthetic devices, petroleum explorations, cancer diagnosis, and tumor treatment.
In order to fully understand the role of solvents in corroles tautomerization and how they can influence its absorptive and electrochemical properties, we investigated the photophysical, electrochemical, and spectroelectrochemical properties of free-base triphenylcorrole (H3TPCor). We characterized corroles light absorbing behavior in various solvents across various concentrations and we also characterized the photophysical properties in its reduced & oxidized states and how solvents can affect their stability. We have analyzed our experimental results and we have developed a mechanism to show solvent effect in the tautomerization reaction of H3TPCor. UV-Visible data shows a trend indicating that the transition from tautomer 1 to tautomer 2 is dominated by the H-bonding donating ability of the solvent molecules with the core of H3TPCor. Tautomer 1 shows four oxidation potentials in the electrochemistry experiments, while tautomer 2 shows three oxidation potentials. Both tautomers have only one reduction potential. UV-Visible spectra of reduced H3TPCor species all followed the same trend and the spectra shifts into a shape that resembles the spectrum of the second tautomeric form indicating that the reduced species prefers this form irrespective of the solvent used. When oxidized, H3TPCor’s spectrum resembles the spectrum of the first tautomeric form of H3TPCor.
Poster #: 64
Campus: San José State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: vasculature, tissue phantom, ultrasound
Project Title: Fabrication of 3-D Vasculature in Tissue-Mimicking Material Using a Removable Pattern
Author List:
Wong, Sze Chung; Undergraduate, Mechanical Engineering, San José State University, Presenting Author
Choi, Mark; Graduate, Mechanical Engineering, San José State University
Lee, Sang-Joon (John); Mechanical Engineering, San José State University
Abstract: Tissue-mimicking materials (TMMs) serve as functional surrogates for studying the effects of prospective treatments such as compression therapy as well as ablation techniques such as high-intensity focused ultrasound (HIFU). The patterning of vascular cavities in the material further enables observation of interactions with simulated blood flow. The purpose of this study is to develop a method of fabricating tissue phantoms with 3-D vasculature. The tissue phantom is made using a polysaccharide gellan gum, which has been used in prior research as a tissue-mimicking material for HIFU ablation therapy. This material, however, is limited to moderate temperature (~70 °C) and has poor chemical resistance. Therefore, conventional methods of removing intricate patterns from a mold (e.g., melting or chemically dissolving) are generally not viable. Accordingly, a method involving the use of a soft removable pattern is presented in this work. The process involves the following major steps: [1] convert vascular geometry into a mold, [2] fabricate the mold in water-soluble polyvinyl alcohol (PVA) using a 3-D printer, [3] cast a soft polydimethylsiloxane (PDMS) pattern into the PVA mold, [4] dissolve the temporary PVA mold in water, [5] transfer the PDMS pattern to a mold cavity and cast the TMM (gellan gum) around the pattern, [6] remove the PDMS pattern by extracting it from the TMM specimen. Since PDMS and the tissue-mimicking material are both pliable, a complex 3-D vascular network can be made without structural damage to either material. Test specimens with different degrees of geometric complexity are demonstrated using this fabrication process to determine limitations of structural integrity for the gellan gum. The geometric changes include different lumen diameters, branching angles, and multiple generations of branching.
Poster #: 65
Campus: Cal Poly San Luis Obispo
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Microfluidics, Point of Care Diagnostic, Micropad
Project Title: Size Reduction Thresholds of Paper-Based Analytical Devices (µPADs)
Author List:
Strong, E. Brandon; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Martinez, Andres W; Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo
Martinez, Nathaniel W; Biological Sciences, California Polytechnic State University, San Luis Obispo
Abstract: Paper-based analytical devices, also known as microPADs (µPADs), are emerging as cost-effective, reliable, and portable biochemical assays capable of supplying real-time data. µPADs are fabricated via wax printing and utilize capillary action to wick small amounts of fluid through lateral hydrophilic channels. With the appropriate design, µPADs can wick samples through in-series test zones and provide a qualitative assessment of the analyte of interest. While µPAD size is limited by the imprecise nature of the wax printing process, device size may be able to be further reduced using an oxidizing agent following fabrication. The purpose of this investigation is to determine the size reduction thresholds of µPADs in aqueous sodium periodate (NaIO4).
Methods: Standard 20.25cm2 µPADs were saturated in varying concentrations of NaIO4(aq.) for times ranging between 9 and 96 hours. Following the optimization of saturation parameters, minimum hydrophilic channels, fluid required for wicking, and wicking speed were tested. Horseradish peroxidase (HRP) stability was measured. Saturated devices were then viewed under a scanning electron microscope and visually compared to standard µPADs.
Results: The optimal saturation parameters were determined to be 0.5M NaIO4 for 48 hours, resulting in an approximately 78% reduction in device area. Following miniaturization, there were significant reductions in the minimum hydrophilic channel width (~49% smaller), fluid required for wicking (~75% less), and wicking velocity (~47% slower), as compared to standard µPADs. Dried HRP is stabilized for ~7 days over controls, at room temperature.
Conclusion: Saturation in an NaIO4(aq.) solution resulted in significant size reduction of paper-based microfluidic devices. We recommend that devices be saturated in 0.5M NaIO4(aq.) for 48 hours for an optimal balance between cost and time required. Wicking time decreased significantly after saturation, caused potentially by condensed cellulose fibers displayed in SEM images. Reduced wicking time may increase test sensitivity by allowing samples more time to move through test zones, with increased enzyme stability. While wax printing implements size restrictions on µPADs, device area can be further reduced through extended submersion in aqueous sodium periodate. Miniaturized µPADs may enable healthcare professionals to design ultra-portable devices requiring miniscule sample sizes.
Funding: CSUPERB New Investigator & Howell Awards
Poster #: 66
Campus: San Diego State University
Poster Category: Disease (Pathogens)
Keywords: bacterial minicells, tumor inflammation, colon cancer
Project Title: Bacterial minicells delay tumor progression and decrease colon inflammation in a mouse model of colon cancer
Author List:
Tian, Mengxi; Graduate, Biology, San Diego State University, Presenting Author, Eden Award Finalist
Meshgin, Nairika; Undergraduate, Biology, San Diego State University, Presenting Author
Khan, Mohammad; Postdoc, Biology, San Diego State University
Grenier-Davis, Shea; Graduate, Biology, San Diego State University
Tsuji, Shingo; Vaxiion Therapeutics, Inc.
Giacalone, Matthew; Vaxiion Therapeutics Inc.
McGuire, Kathleen; Biology, San Diego State University
Abstract: Engineered bacterial minicells (VAX-IP) are non-replicating, nano-sized bacteria that are capable of targeting specific tumor cell-surface proteins and delivering a membrane pore-forming toxin that rapidly kills the cells. Minicell-based therapeutics are highly effective at tumor stabilization and regression in some animal models, but no study on colorectal cancer (CRC) has been reported. In this study, a colon cancer mouse model is being used to evaluate the therapeutic potential of VAX-IP in CRC. By conditionally-deleting the tumor suppressor Apc gene only in colon, tumors develop but leave the immune system intact. VAX-IP treatment of mice (1.5×109 cells/ml, 1 dose/week) from 8-13 weeks of age, a time when lesions are only in the earliest stages, significantly decreases tumor number in mice harvested at 6 months of age (p=0.04). Similarly, when animals were treated later during the lesion development stage (age 14-19 weeks), a more significant decrease in tumor number (p=0.007) was observed.
Studies have shown that inflammatory mediators significantly contribute to tumor progression in CRC. To study whether VAX-IP might reduce tumor load by modulating the colonic environment and mucosal immunity, Chloracetate Esterase (CAE) stains for inflammatory granulocytes and mast cells were used. Numbers of CAE+ cells in normal or tumor-adjacent tissue were significantly lower in VAX-IP-treated mice in comparison to PBS-treated controls (p=0.0002). Immunohistochemistry staining for inflammatory, myeloid-lineage cells using the marker CD11b also showed significantly decreased numbers in the VAX-IP animals treated at 14-19 weeks of age (p=0.003) but not in 8-13 weeks (p=0.4) or parental control (p=0.6) groups. Because macrophages also express CD11b, the F4/80 marker was analyzed on these mouse tissues, but no significant differences were found. The three stains together suggest the difference in CD11b and CAE positive cells is in granulocytic cells and a granulocyte-specific marker Ly6G is being used to confirm this hypothesis. Together, these data strongly support the conclusion that VAX-IP don’t affect the colon immune environment in the absence of tumors but when tumors are present, VAX-IP reduce inflammatory granulocytic infiltration into the colonic environment, which could contribute to the reduced tumor load in this mouse model of CRC. This work is supported by a Joint-Venture Grant from CSUPERB and NCI grant #1U01CA162147.
Poster #: 67
Campus: CSU Northridge
Poster Category: Disease (Pathogens)
Keywords: Protease inhibitor, Staphylococcus caprae, Microbiome
Project Title: A Protease Inhibitor-Producing Staphylococcus caprae Isolated from the Human Skin Microbiome
Author List:
Rogers, Umu; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Bermudes, David ; Biology, California State University, Northridge
Abstract: Protease inhibitors are widely recognized for their therapeutic utility in treating diseases ranging form high blood pressure to HIV. Protease inhibitors are known to be secreted by some bacteria, but relatively few studies have examined the presence or physiological roles of bacterial secreted protease inhibitors associated with the human microbiome. Protease inhibitors produced by bacteria associated with the human microbiome have the potential to contribute to overall stasis, for example, by limiting proteolytic enzymes of other microbes or the host that break down host proteins and promote inflammation. Protease inhibitors could also contribute to the pathogenesis of opportunistic infections by inhibiting neutrophil elastase that normally contributes to suppression of invading microbes. We initiated a study of protease inhibitor-producing bacteria isolated from normal human subjects by cultivating skin swabs on blood agar plates at 37 C in 3% CO2 in order to isolate a variety of cultivable species. We then assessed these strains for production of trypsin inhibitors using protease inhibitor detection plates. One of the several protease inhibitor-producing species we isolated was Staphylococcus caprae, a coagulase-negative species found on both goats and humans that is associated with bone and joint infections. Using trypsin-bound agarose bead affinity purification from culture supernatants followed by SDS-PAGE we identified three prominent bands of approximately 4, 10 and 14 kDa. Our future studies will include identification of the proteins by MALDI-TOF and examining the range of proteases that they inhibit in addition to trypsin. Our results suggest that protease inhibitor-producing bacteria associated with the human microbiome may be more widespread than previously recognized.
Poster #: 68
Campus: CSU Long Beach
Poster Category: Disease (Pathogens)
Keywords: endodontics, Enterococcus faecalis, biofilms
Project Title: The Bactericidal Effect of Sodium Hypochlorite and Hypochlorous Acid on Enterococcus faecalis Model Oral Biofilms
Author List:
Vinh, Jennifer; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
VanDusen, Austin; VA Long Beach Medical Center
Chan, Stephen; VA Long Beach Medical Center
Davis, Stephen; VA Long Beach Medical Center
Dillon, Jesse; Biological Sciences, California State University, Long Beach
Abstract: Infections found in endodontic practices are commonly caused by an excess growth of bacteria. These bacteria form biofilms, which are often highly resistant to treatment. Although both eradication and irrigation are used to treat oral infections, the latter is more commonly used. Because of its ability to dissolve tissue, sodium hypochlorite (NaOCl) or ‘bleach’ is the most widely used treatment. However, the usage of NaOCl against mature biofilms, can result in damage to patient tissues. Hypochlorous acid (HOCl) is less cytotoxic, however its efficacy against oral biofilms is not known. Enterococcus faecalis are common in endodontic infections due to their ability to thrive in diverse environmental conditions, including nutrient-deprived environments, thus, preventing the usage of less potent irrigants. The purpose of this study is to compare the effect of sodium hypochlorite and hypochlorous acid (HOCl) on a 3-week-old E. faecalis biofilm. E. faecalis was grown anaerobically on hydroxyapatite (HA) discs coated with bovine dermal collagen I for three weeks in order to develop a mature biofilm. These biofilms were treated with either a 0.25% NaOCl or 5% HOCl Carr solution for 10, 20, and 30 seconds. The cells were stained with a live/dead stain (LIVE/DEAD® BacLight™ Bacterial Viability Kit L-7012) and then visualized using a confocal laser scanning microscope (Olympus IX-81). The percentage of live and dead cells were quantified via image analysis software. At the concentrations used, HOCl had kill rates 14.2% and 10.1% higher than NaOCl for 10 and 20 second intervals, but not for the 30 second treatment. The comparable results confirm that HOCl is an effective irrigant for E. faecalis biofilms and could be applied to clinical situations, given its lower cytotoxicity. This research provides important insights in the treatment of recalcitrant oral infections and will enable future diagnostic and treatment approaches.
Poster #: 69
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: HSV-2, liposomes, vaccine
Project Title: Comparative Efficacy of Liposomal Vaccines Containing Different HSV2 gD Epitopes Following Intravaginal HSV2 Challenge of BALB/c Mice
Author List:
Rubio, Jennifer; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author, Eden Award Finalist
Ho, Sam; Molecular Express Inc.
Adler-Moore, Jill; Biological Sciences, California State Polytechnic University, Pomona
Abstract: Introduction: Herpes Simplex Virus 2 (HSV-2) causes genital infection in about 20% of the U.S. population and in 500 million people worldwide. This infection cannot be cured by antiviral drug therapy, underscoring the need for an effective vaccine. Our laboratory has been focused on developing a liposomal HSV-2 vaccine containing three of the most immunogenic epitopes within the viral envelope gD protein: gD 1-23, gD 199-222 and gD 264-285 (PEP1, PEP2, PEP3, respectively). The present study was done to compare the different gD epitopes used alone or in combination in the liposomal vaccine with the adjuvant Monophosphoryl lipid A (MPL).
Methods: Liposomal vaccines with MPL or PBS were administered 3X subcutaneously to inbred BALB/c mice (n=12/group) d0, d28, d56. The liposome groups were as follows: gD3PEP as a single tripeptide; a mixture of PEP1+PEP2 + PEP3; PEP1; PEP1 + PEP2; PEP1 + PEP3, PEP2; PEP2 + PEP3; PEP3. Serum was collected d59 (n=5/group) to assess Neutralizing Antibody Titers and to perform anti-gD epitope IgG isotyping by ELISA. Remaining mice were challenged intravaginally with 10X LD50 HSV2 on d70 and monitored for disease signs (mucosal and neurological signs), weight loss and morbidity 2X/day to d98. Vaginal swabs were collected d72 to assess viral burdens using a Plaque Forming Unit assay.
Results: Liposomes with all three peptides (PEP1+PEP2+PEP3), gD3pep, PEP1, PEP1 + PEP2 or PEP1 +PEP3 enhanced survival (85% to 100%) versus PBS, PEP2 or PEP3 (0% survival) and PEP2 + PEP3 (40% survival). Weight loss and disease signs paralleled survival results. Mice vaccinated with PEP1+PEP2+PEP3 or gD3pep, PEP1 alone, PEP1 + PEP2 or PEP1 + PEP3 had significantly reduced vaginal viral burdens (P<0.0021) and significantly elevated Neutralizing Antibody titers versus controls(P<0.0165). Results from the anti-gD epitope IgG isotyping indicated that prolonged survival was associated with a more pronounced Th2 response given the high concentration of anti-gD epitope IgG1.
Conclusions: PEP1 alone in the liposomes was as effective in protecting BALB/c mice against HSV-2 vaginal challenge as a mixture of the three gD epitopes or the single tripeptide (gD3PEP) in liposomes. In comparison, PEP2 or PEP3 in liposomes were not protective. By using just one epitope, vaccine production would be easier and less expensive although these results need to be examined in an outbred mouse population, more representative of the heterogeneity of the human population.
Poster #: 70
Campus: San Diego State University
Poster Category: Disease (Pathogens)
Keywords:
Project Title: Investigating the role of the LytRS Regulatory System in Group B Streptococcal colonization and disease
Author List:
Alhajjar, Norhan; Undergraduate, Department of Biology and Center for Microbial Sciences, San Diego State University, Presenting Author
Najera, Susana; Undergraduate, Department of Biology and Center for Microbial Sciences, San Diego State University, Presenting Author
Deng, Lewin; Graduate, Department of Biology and Center for Microbial Sciences, San Diego State University
Doran, Kelly S.; Department of Biology and Center for Microbial Sciences, San Diego State University
Abstract: Streptococcus agalactiae (Group B Streptococcus, GBS) is typically a commensal bacterium that colonizes the vaginal tract of healthy women. However, in immune compromised individuals such as newborns, GBS may transition to an invasive pathogen. Despite currently recommended intrapartum antibiotic prophylaxis for GBS-positive mothers, GBS remains the leading cause of neonatal sepsis and meningitis. Little is known about what triggers GBS to switch from asymptomatic colonization to causing invasive disease. One way that bacteria respond to environmental changes is through two-component systems (TCS). TCS typically consist of a membrane-associated sensor kinase and a cytoplasmic response regulator that alters gene expression. We hypothesize that TCSs in GBS play a role in the bacteria’s ability to persist in the host and to cause disease. One TCS that has not been studied is the LytRS system. We examined a mutant GBS strain that lacks the LytR response regulator and observed an increase in invasion into human brain microvascular endothelial cells (hBMEC) by the GBSΔlytR mutant in vitro, which was correlated with an increased risk for development of meningitis. Histopathology of brain tissues of individual mice exhibited increased meningeal thickening and cellular infiltration in mice infected with the ΔlytR mutant. Additionally, using RT-qPCR and ELISA based assays, we show that hBMEC secrete more pro-inflammatory cytokines such as IL-6, IL-8, CXCL1, and IL-1 when infected with the ΔlytR mutant compared to the WT strain. Interestingly, the ΔlytR mutant is cleared rapidly in our murine vaginal colonization model while WT bacteria are able to persist in the mouse vaginal tract. We hypothesize the LytS sensor, which is directly upstream of LytR, and the immediate downstream gene, CstA (putative carbon starvation protein A) that is regulated by LytRS, will contribute to GBS virulence. In current studies we are using targeted allelic mutagenesis to engineer GBS strains lacking either LytS or CstA and will determine the role of these factors to GBS colonization and disease. Understanding the mechanisms of GBS regulation during disease progression may lead to the development of more effective therapies to prevent GBS infections.
Poster #: 71
Campus: San José State University
Poster Category: Disease (Pathogens)
Keywords: mouse, inflammation, motor reflexes
Project Title: The Effects of Lipopolysaccharide Induced Inflammation on Spinal Cord Excitability
Author List:
Sanghera , Puneet; Undergraduate, Biological Sciences, San José State University, Presenting Author
Haile, Mulatwa; Undergraduate, Biological Sciences, San José State University, Presenting Author
Chang, Morgan; Undergraduate, Computer Science, San José State University
Putnam, Shea; Graduate, Biological Sciences, San José State University
Wilkinson, Katherine A.; Biological Sciences, San José State University
Abstract: Peripheral inflammation alters the excitability of dorsal horn interneurons and increases flexor reflex strength (Dubner & Ruda, 1992); however, its effect on the spinal stretch reflex is not well understood. The stretch reflex is a muscle contraction in response to muscle stretch sensed by Group Ia muscle spindle afferents. We hypothesize that lipopolysaccharide (LPS) induced inflammation will increase spinal cord excitability, which is a measure of stretch reflex central nervous system processing. To test this hypothesis, we measured Hoffman’s (H) reflex, the electric analog of the stretch reflex, in adult mice receiving an injection of LPS (.5mg/kg) or saline (200µl). Adult male mice (C57Bl/6) were anesthetized with doses of a solution of Ketamine (100 mg Ketamine per kilogram of body weight) and Xylazine (10 mg per kilogram of body weight). After sedating and exposing the sciatic nerve, stimulating electrodes were placed around the sciatic nerve and recording electrodes were placed between the third and fourth interossi foot muscles. The sciatic nerve was stimulated at current strengths from H-wave threshold (T) to 8T (20 x 0.1 ms pulses at 0.1 Hz or 5 Hz). Using LabChart software, the maximum M wave amplitude (Mmax), maximum H wave amplitude (Hmax), and latencies of both waves were measured. We compared the ratio of the maximal H wave over the maximal M wave (Hmax/Mmax), which reports the percentage of motor neurons activated by electrical stimulation of Group Ia muscle sensory neurons. Increased spinal cord excitability would reflect in a larger Hmax/Mmax. We found that LPS induced inflammation does not alter the Hmax/Mmax percentage of motor neurons activated by electrical stimulation of Group 1a muscle sensory neurons. Additionally, we evaluated rate dependent depression (RDD), a measure of the change in H amplitude of the Hoffman reflex over consecutive stimulations at high frequency (5 Hz). No significant differences in RDD were observed in both saline and LPS groups. Although we found that spinal cord excitability, as measured by Hmax/Mmax and RDD, was not altered by LPS, previous findings suggest that inflammation decreases the sensory response to stretch from the Group Ia muscle spindle afferents. Future studies will determine whether stretch reflex strength is affected following systemic inflammation.
Poster #: 72
Campus: CSU Fullerton
Poster Category: Disease (Pathogens)
Keywords: Essential Oils, Multidrug resistance bacteria, Antibiotic resistance
Project Title: Antimicrobial activity of essential oil blends against a great diversity of bacteria, including ESKAPE and anaerobic pathogens
Author List:
Quinn, Brettni; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Sanchez, Gerardo
Fernandez Caniggia, Liliana ; Hospital Aleman, Buenos Aires, Argentina
Ramirez, Maria Soledad; Biological Science, California State University, Fullerton
Abstract: Due to the emergence of bacteria showing extensive antimicrobial resistance (AMR), we are in danger of entering into the post antibiotic era. Despite many efforts to synthesize and retrieve novel antimicrobials from natural sources, the release of new antibiotics has been scarce in the last years. During this time, extensively-drug and pan-drug resistant bacteria have continued to spread, exposing an urgent need to find alternative strategies to combat them. The use of some phytochemicals, such as essential oils (EOs), could constitute an alternative approach to treat human infections, particularly those caused by emerging multi-drug-resistant isolates. The aim of the present work is to evaluate the antimicrobial activity of EO blends against a great diversity of bacteria.
A total number of fifty-three strains belonging to 28 aerobic and 8 anaerobic species of Gram-negative and Gram-positive bacteria were exposed to sixteen EOs. Moreover, the five independent components of one of the EOs were also tested for their antimicrobial activity. The Disk Diffusion Method (DDM) was used as the first screening method to visualize antibacterial activity. The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentrations (MBC) were also performed.
The DDM showed that 14 out of the 16 EOs exhibited bacterial growth inhibition. The most active EOs (oregano, thyme, wintergreen, Thieves®, tea tree, peppermint) showed an average in the diameter of the inhibition halos of 13 mm for the least active to 74 mm for the most active. The MIC results were in agreement with the DDM results showing the notable activity of the tested EOs against different species. For some of the EOs the MIC value was < 0.0390625% of its original pure activity (100%), exhibiting a strong activity.
The detailed analysis of the different components of the EO in Thieves®, showed that clove and cinnamon bark are the components that exhibit significant antibacterial activity.
Since we have tested a wide variety of species, we can conclude that our results shed light in the potential use of EOs to combat antimicrobial resistance. EOs are promising antibacterial agents that show great magnitude of growth inhibition in clinically relevant species. More studies are needed to continue exploring the impact of our findings as well as the application of EOs as a therapeutic option to treat infections.
Poster #: 73
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: Δ9-tetrahydrocannabinol, immunocompromised, Candida albicans
Project Title: Immunocompromised murine responses to a systemic Candida albicans infection are modulated by chronic ∆9-tetrahydrocannabinol treatment
Author List:
Marentes, Adam ; Graduate, Biology, California State Polytechnic University, Pomona, Presenting Author
Vitharanage, Hansini; Graduate, Biology, California State Polytechnic University, Pomona, Presenting Author
Adler-Moore, Jill; Biology, California State Polytechnic University, Pomona
Buckley, Nancy; Biology, California State Polytechnic University, Pomona
Abstract: Δ9-tetrahydrocannabinol (THC) is known to suppress resistance to bacterial, viral and protozo-an infections. However, the effect of THC on resistance to fungal infections is unclear. Recent-ly, we found that chronic THC treatment decreased resistance to the yeast Candida albicans (C. albicans) in immune competent mice. However, nothing is known about how THC affects re-sistance to a fungal infection in an immunocompromised mouse. Thus, our objective was to assess how chronic THC affects an immunocompromised mouse’s ability to ward off systemic candidiasis in both a memory and acute model of infection. 5-fluorouracil (5-F) is a commonly prescribed anti-cancer drug and is also a potent immunosuppressor, increasing susceptibility to C. albicans infection. In our experiment, c57BL/6 female mice were treated via an intraperito-neal (IP) injection with vehicle (ethanol, cremophor, saline (1:1:18)) or THC in vehicle (16mg/kg) 4 days a week, for three weeks (experimental days 1-18). For the memory response model, on day 2, the mice received a priming dose (0.75×105 cells) of C. albicans. On day 16, mice received an intravenous (IV) injection of 5-F (0.1ml of 50mg/ml). On day 19, mice were infected with an IV injection of 5×105 C. albicans cells. For the acute infection model, the mice only received an IV injection of C. albicans on day 19 without a priming dose. On day 22, tis-sues were harvested from some mice (n=5) to assess cytokine production and tissue fungal load. Remaining mice (n=7) were observed for 2 weeks for survival and morbidity. We found that 5-F treatment severely decreased survival, cell blood count and splenic IL-12p40 and increased kidney fungal load. We also found that in both infection models, mice treated with 5-F and THC succumbed a day earlier to the yeast infection compared to 5-F and vehicle treated mice. We suggest that although THC does make the 5-F treated mice slightly more susceptible to the infection, it is not significant enough to conclude that chronic THC treatment is detrimental to immune compromised mice.
Poster #: 74
Campus: Cal Poly San Luis Obispo
Poster Category: Disease (Pathogens)
Keywords: Urinary tract infection, Escherichia coli, growth curve
Project Title: Friend or foe? A growth-based assay distinguishing commensal from uropathogenic Escherichia coli
Author List:
Wilmer, Abby; Undergraduate, Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, Presenting Author
Leavitt, Kelly; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Yep, Alejandra; Biological Sciences, California Polytechnic State University, San Luis Obispo
Abstract: Urinary tract infections (UTIs) account for nearly 25% of all bacterial infections, a significant public health concern. Uropathogenic Escherichia coli (UPEC) is responsible for 80% of uncomplicated UTIs. The majority of E. coli, including UPEC, reside harmlessly in the lower digestive tract, but when UPEC reaches the urethra, it can colonize the bladder. Since increasing rates of antibiotic resistance complicate UTI treatment, studies that focus on understanding transmission of UPEC from the gut to the urinary tract are crucial for determining preventive measures. A main hurdle in these studies has been the difficulty in identifying an E. coli isolate as commensal or UPEC, since there are no consistent genetic markers. Our research aims to establish an affordable, quick, growth-based assay that will differentiate UPEC from commensal strains by developing a specialized growth media in which UPEC and commensal strains behave significantly different. Ten fecal strains, ten UPEC strains collected from patients with uncomplicated UTIs, as well as model UPEC strain CFT073 were tested in a series of conditions to assess the separation in growth. Four technical replicates of each strain were used to inoculate 96-well plates containing specialized media. OD600 was measured over a 12-hour period using a microplate reader. There was no significant growth variation in MOPS minimal medium with iron, but when using MOPS no iron, growth separation was observed. In M9 minimal medium, 0.2% dextrose, UPEC outgrows commensal strains at pH 5.5 and 7.5, but the difference disappears at pH 8. Urine contains small concentrations of peptides that UPEC consumes as an energy source, so to simulate a urine-like environment, non-carbohydrate carbon sources were tested. Tryptone and alanine allowed for differential growth at pH 6.55 and 5.6 in M9, respectively. Ongoing experiments will test the effect on growth of commensal and UPEC strains when human lipocalin 2 is added into solution. Lipocalin 2 is a neutrophil-secreted protein that binds to the siderophore enterobactin (Flo TH, et al. 2004. Nature 432:917-912). Our results thus far indicate a growth-based assay can be used to separate commensal from UPEC isolates. This research is supported by a Center for Applications in Biotechnology (CAB) grant at Cal Poly, San Luis Obispo (AY).
Poster #: 75
Campus: CSU San Marcos
Poster Category: Disease (Pathogens)
Keywords:
Project Title: Restoration of T cells in the Epithelial Layer of the Small Intestine in Obese Mice
Author List:
Delgadillo Miranda, Nadia; Undergraduate, Biology, California State University San Marcos, Presenting Author
Bshara, Savannah; Undergraduate, Biology, California State University San Marcos
Park, Christa; Undergraduate, Biology, California State University San Marcos
Jameson, Julie; Biology, California State University San Marcos
Abstract: Obesity and type 2 diabetes are an increasing epidemic in the United States. Studies show that obesity causes complications such as barrier dysfunction and chronic non-healing wounds. Our laboratory has demonstrated that obesity and type 2 diabetes negatively affects the function and number of T cells in the epithelial layer of the small intestine. However, little is known about whether these T cells can be restored in number. In this study, we investigated whether diet-induced weight loss would restore T cells in the small intestine of obese mice. To test this hypothesis, mice were separated into three cohorts. The control lean cohort consumed a 10% kcal Normal Chow Diet (NCD) for 14 weeks. The obese cohort consumed a 60% kcal high fat diet (HFD) for 14 weeks and the diet-induced weight loss cohort consumed a 60% kcal HFD for 7 weeks followed by a 10% kcal diet for the remaining 7 weeks. The small intestine was examined by snap freezing the tissue, cutting 5um sections with a cryostat machine, staining the tissue sections with antibodies specific for T cells, and examining the sections under an immunofluorescent microscope. Images were examined using Photoshop and ImageJ to quantify the number of T cells per villi in the small intestine. Here we demonstrate that diet-induced weight loss restored T cell numbers to normal levels in the small intestine. In addition, splenocytes were also analyzed to give a systemic outlook of all T cells traveling in the body. T cells isolated from the spleen were quantified and analyzed using a flow cytometer and FlowJo software. Interestingly, we discovered that the number of T cells in the spleen of the dieted mice was not restored to normal control levels. This suggests that dieting improves T cell numbers in barrier tissues, but not the spleen. We hypothesize that the lymphocytes in the spleen have either migrated out, reduced their proliferation or undergone apoptosis. Together our studies show that losing weight by short term dieting can improve some immune cell parameters, while others remain compromised.
Funding was provided by CSUPERB Presidents’ Commission Scholars Program.
Poster #: 76
Campus: San Diego State University
Poster Category: Disease (Pathogens)
Keywords:
Project Title: Establishing in vitro and in vivo models of vaginal colonization by Staphylococcus aureus
Author List:
Deng, Liwen; Graduate, Department of Biology and Center for Microbial Sciences, San Diego State University, Presenting Author
Kwiecinski, Jakub; Department of Microbiology, University of Iowa Carver College of Medicine
Horswill, Alexander R.; Department of Microbiology, University of Iowa Carver College of Medicine
Doran, Kelly S.; Department of Biology and Center for Microbial Sciences, San Diego State University
Abstract: Staphylococcus aureus is an important pathogen responsible for nosocomial and community aquired infections in humans, and cases of community associated methicillin- resistant S. aureus (CA-MRSA) infection have continued to increase despite widespread preventative measure. Reports have suggested an increase in CA-MRSA infections in pregnant and post-partum women as well as outbreaks in newborn nurseries. S. aureus has been reported to colonize the vagina in up to 22% of pregnant women, however little is known about the specific bacterial factors that promote vaginal colonization and subsequent infection. We hypothesize that S. aureus must express factors that mediate interaction with vaginal epithelium and compete or cooperate with other common colonizers in order to promote niche establishment. To investigate how S. aureus is able to interact with vaginal epithelial cells and persist in the vaginal tract, we have adapted models of Group B Streptococcal vaginal colonization. Using a mouse model of vaginal colonization, we have demonstrated that both invasive and colonizing MRSA isolates, CA-MRSA USA300 and MRSA-252, persist in the murine vaginal tract. Further, we have also shown that USA300 attaches to human vaginal epithelial cells (HVEC) in vitro. We have previously established that fibrinogen (Fg) is an important vaginal matrix component for bacterial colonization, thus we sought to investigate the importance of MRSA-Fg interactions for vaginal cell interactions. We have constructed single and combination mutants of Fg adhesins, including ClfA, ClfB, FnbpA, FnbpB, and SdrCDE and have observed that inactivation of multiple adhesins reduced Fg binding and the ability to adhere to HVEC compared to the parental strain. Future studies aim to utilize these in vitro and in vivo models of vaginal colonization as well as our adhesin mutants to determine the importance of Fg binding to MRSA vaginal colonization and polymicrobial interactions within the vaginal tract.
Poster #: 77
Campus: CSU San Bernardino
Poster Category: Disease (Pathogens)
Keywords: microbiome, Dcy , Drosophila
Project Title: Gut Bacteria Supplements – Best Frenemies?
Author List:
Grayson, Kaitlyn; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Bournias-Vardiabasis, Nicole; Biology, California State University, San Bernardino
Abstract: Microbiota in the gut have a symbiotic relationship with their host organisms. Some aid in digestion and the breakdown of nutrients and others can help regulate intestinal physiology. However, others may not be as beneficial and feed on the epithelial lining of the gut, which can lead to inflammation. This field of study is relatively new and the effects of microbiota are not fully understood. Previous studies have concentrated on measuring diversity within the microbiome, ratios of different species, and interspecies interactions. In Drosophila, previous studies have shown a relatively low diversity in the gut. In this study, we decided to use probiotic cultures and alter bacterial ratios to identify the effects that various species may have on overall fly health. For this study, two strains of Drosophila melanogaster were used: Dcy KO and Dcy rescue. Dcy KO flies are a strain that is unable to produce the Dcy protein, which is an important structural component of the peritrophic matrix. Due to this deficiency, Dcy KO flies have a leaky gut, which makes them more susceptible to inflammatory disease. Within these two main groups, there were four subgroups (depending on the microbes used) with 75 male individuals each: control, A. fumigatus, S. marcescens, and L. plantarum. The control group were fed standard cornmeal-sucrose medium. The other three were provided medium inoculated with 50 µL of bacterial or fungal suspension. The lifespan observation made indicated that S. marcescens reduced the lifespans of both genotypes by an average of one week. A. fumigatus exposure resulted in a very slight decrease in the Dcy KO populations’ lifespans but it was not statistically significant. L. plantarum exposure didn’t have any significant effect. Heightening the insects’ exposure to L. plantarum probably did not have a significant effect because this is the predominant species found within Drosophila’s gut microbiome and it simply filled up other niches while continuing regular functions. A. fumigatus most likely didn’t have a significant effect because yeast cells are larger than bacterial cells and therefore could not penetrate the peritrophic matrix. Future studies will include the addition of a nutritional supplement to determine if the altered bacterial ratios affect nutrient absorption. In addition, a courtship assay will be performed to further understanding on how changes in the microbiome affect behavior patterns.
Poster #: 78
Campus: Sacramento State University
Poster Category: Disease (Pathogens)
Keywords: Biofilm, Neuroendocrine, Metatranscriptome
Project Title: Adrenergic Receptor Antagonists Mediate Differential Transcriptomic Responses During Neuroendocrine Crosstalk with the Skin Microbiome
Author List:
Fuentes, Jaime; Graduate, Biological Sciences, California State University, Sacramento, Presenting Author
Singh, Shaleni; Undergraduate, Biological Sciences, California State University, Sacramento
Crawford, Robert; Biological Sciences, California State University, Sacramento
Abstract: The treatment of chronic skin wounds is complicated by aggregations of biofilm-embedded species of the resident skin microbiota. Biofilm formation occurs through a highly regulated series of transcriptomic alterations that culminate in the production of a self-initiated extracellular matrix (ECM), and these population-dense communities provide resistance for a variety of microbes against immune system mediators and antibiotics in numerous clinical settings. Thus, a critical barrier to wound healing is the paucity of non-antibiotic therapeutics that inhibit microbial growth and prevent biofilm development. Prior work from our lab demonstrated that growth rates and biofilm biomasses of prominent skin species increased significantly by a serum-independent mechanism in the presence of physiological levels of the catecholamine (CAT) neuroendocrine transmitter epinephrine (EPI). We hypothesize here that EPI-mediated conversions to pathogenic phenotypes associated with wound chronicity can be reversed by adrenergic receptor antagonists that will have differential effect on gene transcription. Our data suggests that the β-blocker timolol ameliorates EPI-induced increases in growth and biofilm formation by Staphylococcus aureus and S. epidermidis. We use quantitative Real-Time PCR and a combination of crystal violet staining and Confocal Laser Scanning Microscopy for ECM-bound sugars to demonstrate that EPI regulates an augmentation in gene expression for determinants associated with microbial attachment to collagen during the initial 4 hours of biofilm development. Intriguingly, the EPI-mediated changes to transcriptomic profiles and binding patterns that ultimately increase biofilm biomass were completely abrogated by the addition of timolol. Metatranscriptomic analysis using Next Generation Sequencing Technologies revealed differential, species-specific expression profiles for planktonic and biofilm-associated bacteria. Collectively, these data show that CAT-mediated enhancements to growth and biofilm by species of skin microbiota can be reversed with specific β-blockers and thereby suggest a novel non-antibiotic therapeutic: translational CAT bio-inhibitors to prevent microbial persistence in chronic wounds. These studies were supported by a CSUS undergraduate research experience award.
Poster #: 79
Campus: CSU Fresno
Poster Category: Disease (Pathogens)
Keywords: American Crow, Microbiome, resistome
Project Title: The microbiome and resistome of the American Crow across the urban-rural gradient
Author List:
Nelson, R. Lee; Graduate, Biology, California State University, Fresno, Presenting Author
Castro, Michael; Undergraduate, Biology, California State University, Fresno, Presenting Author
Van Laar, Tricia; Biology, California State University, Fresno
Abstract: The Centers for Disease Control and Prevention (CDC) estimates that over 2 million people per year acquire antibiotic resistant infections, with a mortality of approximately 23,000 annually. Needless to say, antibiotic resistance is a cause for concern. Antibiotic resistance has been studied in abiotic systems such as rivers and fish farms as well as biotic reservoirs like gulls and rodents.
This study focuses on the biotic reservoir Corvus branchyrhyncos, the American Crow. Crows are well adapted to living closely with humans in urban and rural areas. These birds are well-known scavengers which gives them ample opportunity to incorporate drug-resistant bacteria into their microbiome through consumption of contaminated materials. These bacteria can then be deposited into areas of human habitation by defecating birds, particularly during roosting season when their numbers can reach well into the hundreds.
We hypothesize that urban crow populations will display a higher incidence of antibiotic resistance and pathogenic bacteria compared to rural populations.
Fecal samples were collected from the ground below roosting crows and the DNA was extracted using the MoBio Soil Kit and sent to Mr. DNA for 16s rDNA sequencing. Since all bacteria have the 16s ribosomal subunit we use these sequences to identify the organisms. The presence of antibiotic resistance is the genomes of fecal microbes will determined by selective digestion of the remaining DNA followed by ligation of fragments between 2-5 kb into plasmids. The plasmids will be transformed into E. coli and grown in the presence of various antibiotics. If the E. coli are capable of growth, we will know that the presence of resistance to that particular antibiotic was present in the original bacterial population.
Through preliminary culture studies, we have identified multiple antibiotics to which microbes isolated from crow feces are resistant. These drugs include vancomycin, meropenem and hygromycin among others. Based on 16s DNA sequencing of isolated bacteria, we have identified members of many genera, some capable of causing human disease, including members of Enterococcus, Staphylococcus, Bacillus, Klebsiella and Paenibacillus.
We plan to collect samples from more California locations such as the coast, mountains, and deserts. This will give us a more complete understanding of the American crow’s fecal microbiome.
Poster #: 80
Campus: CSU Fullerton
Poster Category: Disease (Pathogens)
Keywords: Parasites, Mechanosensation, CRISPR/Cas9
Project Title: Life under stress: Role of mechanosensitive channels in the human parasite Trypanosoma cruzi
Author List:
Dave, Noopur; Graduate, Biological Science, California State University, Fullerton, Presenting Author, Eden Award Finalist
Wong, Bonnie; Undergraduate, Biological Science, California State University, Fullerton
Cetiner, Ugur; Undergraduate, Department of Biology, University of Maryland, College Park
Sukharev, Sergei; Department of Biology, University of Maryland, College Park
Jimenez, Veronica; Center for Applied Biotechnology Studies and Department of Biological Science, California State University, Fullerton
Abstract: Chagas disease is a tropical parasitic disease caused by the protozoan Trypanosoma cruzi. During its life cycle, T. cruzi must endure various environmental changes as it propagates between an insect vector and a vertebrate mammalian host. These include changes in osmolarity, pH and host conditions. Although the mechanisms used by the parasite to respond to environmental changes have been partially described, the molecular machinery able to sense such fluctuations is still unknown.
In silico analysis of the T. cruzi genome revealed the presence of sensory proteins including a bacterial-like mechanosensitive channel, TcMcS. Mechanosensitive channels are ion channels activated by stretch or tension that is applied to the plasma membrane. Previous studies have shown that mechanosensitive channels transduce signaling pathways that lead to osmoregulation and an increase in virulence factors in pathogenic bacteria, suggesting that TcMcS may have a similar role in T. cruzi. We have developed cell lines in which TcMcS expression levels have been modified in order to assess its role in key physiological functions including osmoregulation and infectivity.
Electrophysiological patch-clamp studies revealed the activation of TcMcS as pressure dependent, confirming that TcMcS is a mechanosensitive channel and could be a permeation pathway for calcium and osmotic osmolytes. Immunofluorescence assays showed a differential localization of the protein in the main life stages of the parasite, suggesting stage-dependent functions. CRISPR/Cas9 knockdown and knockout parasites showed significant defects in growth indicating that TcMcS is an essential protein. Furthermore, under hyposmotic stress, the ability of the parasite to regulate the cell volume was dependent on TcMcS expression levels. Lastly, TcMcS knockdown and knockout parasites show significant defects in metacyclogenesis, as well as, host-cell invasion and intracellular replication.
Overall, this study shows that TcMcS is crucial for T. cruzi osmoregulation and host-cell infectivity. TcMcS is not present in mammalian cells and could represent a suitable drug target for development of new therapeutic alternatives against Chagas disease.
Funding: AHA 16GRNT30280014, CSUF RSCA, CSUPERB New Investigator Grant to VJ.
Poster #: 81
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila, behavior, genetics
Project Title: Neuropeptide F is a Target of Developmental Ethanol Exposure in Drosophila melanogaster
Author List:
Guevara, Amanda; Graduate, Biological Sciences, San José State University, Presenting Author
Gates, Hillary; Undergraduate, Biological Sciences, San José State University
French, Rachael; Biological Sciences, San José State University
Abstract: Fetal Alcohol Spectrum Disorder (FASD) describes a collection of physical and neurobehavioral disorders all of which are due to developmental alcohol exposure (DAE). In both flies and mammals DAE results in a variety of phenotypes; specifically developmental delay, decreased survival, and neurobehavioral changes, including feeding abnormalities and sleep disorders. Despite decades of research on DAE, alcohol’s effects on molecular and neurological pathways are poorly understood. Furthermore, public health campaigns against alcohol consumption during pregnancy have reached the limits of their effectiveness, and the proportion of women that consume alcohol while pregnant has remained steady for the last 15 years. Therefore, it is imperative that we have a greater understanding of the mechanisms underlying ethanol’s deleterious effects during development in order to investigate potential treatments for FASD.
We have established Drosophila melanogaster as a genetic model organism to study FASD. Here, we use this model to examine the effects of DAE on feeding behavior and the expression and function of Neuropeptide F (NPF) following DAE. NPF is a peptide hormone that mediates reward, and that is homologous to mammalian Neuropeptide Y (NPY). NPF/NPY regulates several reward-driven behaviors, including feeding and courtship, and is considered to be a “hunger” signal.
Using spectrophotometric analysis of dyed food to measure food intake, we find that ethanol-reared flies eat less than control flies, consistent with the effects seen in mammals with FASD. In addition, ethanol-reared flies show reduced motivation to eat, as measured by the time that elapses between feeding bouts when flies have free access to food. In addition, our preliminary data indicate that NPF expression is increased in the brains of ethanol-reared larvae, and that partial loss of function of NPF enhances the feeding deficits seen in ethanol-reared flies. Most strikingly, we find that flies with a null mutation in the NPF receptor (NPFR1) display drastically reduced survival when reared in ethanol compared to control animals. This result is unexpected, as there are no published data indicating a survival requirement for NPF signaling in any species. We have found that the critical period for NPFR1 in ethanol-reared flies is during larval development, and we are now investigating the possibility that the animals die of starvation due to insufficient neurochemical motivation to feed.
Poster #: 82
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: MAGP2, integrin, proteolytic cleavage
Project Title: Determining the regulatory role of enzymatic cleavage on MAGP2-induced integrin activation
Author List:
Perez, Aldwin Apollo ; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Miyamoto, Alison; Biological Science, California State University, Fullerton
Abstract: Microfibril-associated glycoprotein 2 (MAGP2) is a 23 kDa protein secreted into the extracellular matrix (ECM) that has both cell and matrix-associated functions. MAGP2 binds to microfibrils in vivo in both non-elastic and elastic-tissues, and stimulates elastic fiber formation in vitro. Enzymatic cleavage of MAGP2 has previously been shown to promote matrix-association. Proprotein convertases (PCs) split MAGP2 into two fragments via a RLRR consensus site: a 20 amino acid C-terminal fragment and a large N-terminal (NT) fragment containing a RGD motif. The RGD motif serves as a recognition site for αvβ3 integrin signaling activation. Activation of αvβ3 receptors via MAGP2 has been shown to be correlated with advanced stage ovarian cancer. However, the role that cleavage of MAGP2 plays in integrin activation has not yet been assessed. I hypothesize that PC cleavage of MAGP2 serves as a potential modulator for integrin signaling. Two mutant constructs of MAGP2 have been used in a preliminary study to determine the role of MAGP2 cleavage in αvβ3 activation. One mutant is a deletion of the last 20 amino acids of MAGP2 thus mimicking a cleaved NT fragment. The other mutant resembles an uncleaved MAGP2 protein made by blocking cleavage through two amino acid changes in the RLRR sequence. Both mutants have been tested for integrin activation using immunoblots of phosphorylated focal adhesion kinase in αvβ3-positive MG-63 cells. Preliminary results from the integrin activation assay suggest the uncleaved MAGP2 mutant potentially decreases integrin activation versus both wildtype and the cleaved MAGP2 mutant in MG-63 cells. Additional preliminary results show a potential role of the C-terminal fragment as a negative regulator of MAGP2-induced integrin activation. Further optimization of the assay is currently being undertaken to determine substrate-level modulation of integrin signaling. Then a PC inhibitor will be used as a complementary system to assess the role of cleavage at an enzyme level. Using the integrin activation assay on both substrate and enzyme will provide clarity on the role PC cleavage has as a post-translational regulator of MAGP2 cell-associated functions. Assessing the role of cleavage on MAGP2-integrin activation will promote understanding of MAGP2-induced ovarian tumor formation via αvβ3-linked angiogenesis.
Poster #: 83
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: RCAN1, Apoptosis, CRISPR-Cas9
Project Title: Analysis of RCAN1 gene knockdown in glucocorticoid-induced apoptosis of human lymphoblastic leukemia cells
Author List:
Hurtado, Joanna; Undergraduate, Biology, California State University, Northridge, Presenting Author
Sanchez, Evelyn; Undergraduate, Biololgy, California State University, Northridge, Presenting Author
Medh, Rheem; Biology, California State University, Northridge
Abstract: Glucocorticoids (GCs) are antileukemic agents that induce apoptosis in leukemic T and B cells, primarily by regulating gene expression. Our laboratory is studying the molecular basis of GC induced apoptosis and GC resistance using CCRF-CEM sister cell lines derived from human acute lymphoblastic leukemia (ALL). We have shown that the synthetic GC dexamethasone (Dex) regulates separate sets of genes in the two sister cell lines: GC-sensitive CEMC7-14 and GC-resistant CEMC1-15. Upregulation of the RCAN1 gene, a Down Syndrome Critical Region Gene, correlates with sensitivity to Dex. Studies presented here aimed to knock out RCAN1 expression to understand its role in lymphoid cell apoptosis. We hypothesize that if GC-evoked upregulation of RCAN1 facilitates apoptosis, knockout of the protein function of RCAN1 will reduce the sensitivity of CEMC7-14 cells to Dex-induced apoptosis. The CRISPR-Cas9-based genome-editing approach was used to successfully knockdown RCAN1 expression in CEMC7-14 cells. Two clones with separate mutations were isolated and the mutations were verified by DNA sequencing. The extent of RCAN1 knockdown was analyzed by Western Blotting, using an antibody specific for the C-terminal region of RCAN1. The results demonstrated a significantly reduced expression of basal as well as Dex-induced RCAN1 protein, but not a complete knockout. MTT assays were performed to assess the effect of RCAN1 knockdown on sensitivity to Dex-evoked apoptosis. The results suggested that RCAN1 knockdown did not significantly protect against Dex-evoked apoptosis. We conclude that either minimal expression of RCAN1 was sufficient to sensitize CEM cells to GC-evoked apoptosis, or that since multiple parallel pro-apoptotic pathways are activated by GCs in CEM cells, knockdown of RCAN1 alone was not sufficient to protect cells from Dex-induced apoptosis. Further studies will utilize RCAN1 knockdown lines to understand RCAN1-downstream gene regulatory events and crosstalk between RCAN1 and other apoptotic pathways.
Acknowledgements: This research is funded by a NIH SCORE SC3 award to RDM (GM081099) and a MARC-U*STAR scholarship to JH. The authors acknowledge the support of the CSUN College of Science and Mathematics and CSUPERB.
Poster #: 84
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Sinorhizobium meliloti, Molecular Biology, Polysaccharide analysis
Project Title: Investigating the Role of ExoS/ChvI Signaling in Polysaccharide Production by Sinorhizobium meliloti
Author List:
Bayz, Kalthoum; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Chen, Esther; Biological Science, California State University, Fullerton
Abstract: Sinorhizobium meliloti is a gram-negative alpha-proteobacterium that uses a two-component regulatory system, ExoS/ChvI, to form a nitrogen-fixing symbiosis with legume host plants. This symbiosis is a model for studying the mechanisms used by invasive bacteria during host interactions and is important for improving agriculture, since fixed nitrogen is often a limiting nutrient for plant growth. An exchange of chemical signals between host and bacteria allows the bacteria to invade root hairs and nodules through host-derived structures called infection threads. ExoS/ChvI influences symbiosis as well as free-living phenotypes including biofilm formation, motility, nutrient utilization, and cell envelope integrity. It is also well-established that S. meliloti exoS/chvI mutants are defective in the production of succinoglycan, an exopolysaccharide required for successful infection of host plants. We investigated whether exoS/chvI mutants are defective in producing other polysaccharides, lipopolysaccharide (LPS) and cyclic beta-glucans, which also have important roles in establishing the symbiosis. LPS and cyclic beta-glucans are important for host invasion and are located in the outer membrane or periplasm, respectively. Using SDS-PAGE and silver staining, we compared the smooth and rough LPS profiles of the chvIK214T partial loss of function mutant with wild-type and LPS mutant controls. We found that the LPS profile of the chvI mutant was indistinguishable from that of wild-type. Using thin layer chromatography, we compared cyclic beta-glucans produced by the chvIK214T mutant with wild-type, and we found that the cyclic beta-glucan of the chvI mutant was indistinguishable from that of wild-type. Thus, our results indicate that ExoS/ChvI does not affect the production of LPS and cyclic beta-glucans. Future work will use a newly-constructed chvI knockout strain to test whether complete loss of function in chvI results in a defect in LPS or cyclic beta-glucan production. This project was supported by the CSU Special Fund for Research, Scholarship, and Creative Activity and by CSUF Biology Research funds.
Poster #: 85
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: fluorescence in situ hybridization, lactase, oral
Project Title: in situ function of uncultivated Candidatus Saccharibacteria lactase
Author List:
Lin, Danny; Undergraduate, Biological Science, San José State University, Presenting Author
Belete, Canaan; Undergraduate, Biological Science, San José State University, Presenting Author
Ghadiri, Farsheed; Graduate, Biological Science, San José State University
Abrams, Michael; Graduate, Biological Science, San José State University
Ouverney, Cleber; Biological Science, San José State University
Abstract: Adult periodontitis, an inflammatory oral disease, may cause the loss of teeth due to sub-gingival plaque. Loss of teeth and degradation of oral health is partly due to plaque buildup on the tooth. Plaque, a term for bio-film specific to the mouth, contains multiple bacterial species. Bacteria metabolize through anaerobic respiration, creating acidic products. These acidic products can degrade the tooth and gum, causing oral disease. Candidatus Saccharibacteria (TM7) is a bacterial phylum that has been associated with adult periodontitis. Characterization of TM7 and its relation to oral disease can contribute to the understanding of oral community interactions. A metagenomic study approach was used to help analyze large genomic DNA fragments from oral communities known to contain TM7. High-throughput DNA sequencing helped identifies a lactase gene within TM7. Two approaches were used to help characterize the novel lactase gene: Fluorescence in situ hybridization (FISH) on oral TM7 bacteria and in vivo gene expression in Escherichia coli (E.coli). Since lactase by itself cannot be detected by the naked eye, Fluorescein Di-β-D-Galactopyranoside (FDG) was used. FDG is a lactose analog that, when hydrolyzed, can fluoresce. This can be detected using Confocal Scanning Microscopy (CSM). E. coli expressing the lactase gene was used as a positive control. Based on the CSM results, we inferred that TM7 can break down lactose within the cell. This demonstrates that TM7 can activate its lactase gene and may play a role in lactose degradation in oral plaque, which may be involved in tooth decay.
Poster #: 86
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: ectoderm, cadherin, neural crest
Project Title: E-cadherin orchestrates neuroectodermal cell fate choices
Author List:
Sorrells-Smith, Lisa; Undergraduate, Biology, California State University, Northridge, Presenting Author
Rogers, Crystal; Biology, California State University, Northridge
Abstract: Early developmental processes are tightly regulated such that successful embryonic development requires tissues to segregate into three germ layers: ectoderm, mesoderm and endoderm during gastrulation. After these tissues are specified, each tissue is further determined, eventually differentiating into all the cells and tissues that will make up the adult organism. We are interested in the process that controls the specification and differentiation of ectodermal tissues. Ectodermal cells will become the neural tube (central nervous system), non-neural ectoderm (epidermis and placodes) and neural crest cells (craniofacial cartilage and bone). Very little is known about the processes that regulate the cell fate choices of ectodermal derivatives, however, previous studies have shown that cell adhesion through cadherin proteins is important. E-cadherin (Ecad) is a calcium dependent cell-cell adhesion molecule, and its downregulation is necessary for cell migration in metastatic cancer cells, but until now its influence on embryonic ectodermal-derivative cell-fate specification has not been recognized. We have performed loss of function experiments in chick embryos and have identified that Ecad is necessary for normal separation of the three ectodermal deriva-tives. Microinjection of CRISPR-Cas9 constructs followed by whole mount immunohistochemistry produced embryos with abnormal ectodermal derivative specification. In vivo Ecad knockdown resulted in a dorsal expansion of neural tube cells marked by Sox2 at the expense of neural crest cells marked by Sox9 and Pax7. These results expand our knowledge of the role of Ecad in early development by showing that it influences early tissue specification. Recognition of the expanded role that Ecad plays may have implications for other aspects of development that involve Ecad such as gastrulation, neural crest migration and the formation of the heart and gut.
Poster #: 87
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: diabetes, microRNA, cell proliferation
Project Title: MicroRNA-375 controls pancreatic alpha and beta cell growth in opposing ways
Author List:
Diaz, Pablo; Graduate, Biology, California State University, Chico, Presenting Author
French, Melissa ; Undergraduate, Biology, California State University, Chico, Presenting Author
Prator, Grace; Undergraduate, Biology, California State University, Chico
Keller, David; Biology, California State University, Chico
Abstract: MicroRNA 375 (miR-375) is a small non-coding RNA that regulates hormone secretion in pancreatic islets and controls the development of pancreatic alpha cells and beta cells. Alpha cells secrete the hormone glucagon which raises blood glucose during times of fasting, and beta cells secrete the hormone insulin which reduces blood sugar after a meal. However, in type II diabetes the overexpression of miR-375 is correlated with reduction in beta cells and an increase in alpha cells which probably contributes to the pathogenesis of the disease. We hypothesize that miR-375 controls cell metabolism and proliferation, but does so differently in alpha cells and beta cells. We believe that miR-375 will have a positive role in cell metabolism and expression of cell growth genes in alpha cells, but will have a negative role in beta cells. To investigate this, we perform cell proliferation assays to monitor the effects of miR-375 inhibition within alpha cells and beta cells. We also perform quantitative polymerase chain reaction (qPCR) to monitor target genes of miR-375 known to play a role in cell growth. Our results indicate that the inhibition of miR-375 has opposite effects within the two cell lines. When transfected with a miR-375 inhibitor, beta cells exhibit increased proliferation and alpha cells exhibit decreased proliferation. This affect might be due to miR-375 target genes which repress cell growth, because they are down-regulated in alpha cells compared to beta cells as gauged by qPCR. Our data supports the model that miR-375 stimulates metabolism and proliferation in alpha cells but inhibits it in beta cells and provides insight into the cellular and molecular mechanisms of type II diabetes.
Poster #: 88
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila, courtship, behavior
Project Title: Characterization of a novel gene required for Drosophila melanogaster mating
Author List:
Zaki, Sadaf; Graduate, Biological Sciences, San José State University, Presenting Author
Sandhu, Jaspal; Undergraduate, Biological Sciences, San José State University, Presenting Author
Luu, Peter; Graduate, Biological Sciences, San José State University
French, Rachael; Biological Sciences, San José State University
Abstract: The common fruit fly Drosophila melanogaster is an ideal model organism to study genetic regulation of innate behaviors. Male Drosophila carry out a complex courtship ritual involving a specific sequence of steps, which depend on the processing of sensory cues by the central nervous system (CNS). The zinc-finger transcription factor-encoding gene fruitless (fru) is the master regulatory gene that controls behavioral sex determination (and thus all sex-specific behaviors) in flies; the male-specific splice form of fruitless (fruM) is both necessary and sufficient for all aspects of the courtship ritual.
We have identified a gene, Trapped in endoderm 1 (Tre1), that is essential for normal courtship behavior in male flies. Tre1 encodes an orphan G-protein-coupled receptor (GPCR) that is required for both germ cell migration and establishment of cell polarity, but has not previously been implicated in courtship. The goal of this research is to characterize the role of Tre1 in Drosophila mating behavior. We found that male flies in which Tre1-expressing cells are feminized through expression of the female-specific isoform of the splicing factor Transformer (TraF) display unusually rapid courtship initiation, and that this phenotype is recapitulated in flies carrying a loss-of-function mutation in Tre1 (Tre1EP496). This phenotype is rare among Drosophila courtship mutants; in the vast majority of cases, feminization of neurons controlling courtship behavior leads to reduced courtship speed and other obvious courtship deficits. Our results therefore suggest that a subset of male-specific neurons in flies function to reduce the speed of courtship initiation. Using confocal microscopy, we found that Tre1-GAL4 is expressed in the olfactory organs and olfactory processing centers of the male nervous system, with little to no expression in these regions of the female nervous system, and this expression pattern was lost in feminized male brains.
Finally, we used neuron-specific RNA interference (RNAi) to confirm that the requirement for Tre1 in courtship is specific to neurons. These data collectively indicate that the Tre1 neurons are required to slow down the courtship ritual, as well as suggest a role for Tre1 in the processing and integration of sensory signals during courtship. We hypothesize that the Tre1 GPCR is involved in sex-specific neuronal pathfinding, and that disruption of this pathfinding leads to improper neuronal connectivity.
Poster #: 89
Campus: CSU Long Beach
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Macrophages, Complement, Inflammasome
Project Title: Complement Protein C1q Suppresses Macrophage Inflammasome Activation During Clearance of Modified LDL
Author List:
Manughian-Peter, Ayla; Graduate, Biological Sciences, California State University, Long Beach, Presenting Author, Eden Award Finalist
Ho, Minh-Minh; Graduate, Biological Sciences, California State University, Long Beach
Fraser, Deborah; Biological Sciences, California State University, Long Beach
Abstract: Cardiovascular disease is the number one cause of death worldwide. Atherosclerosis, a condition characterized by the development of fatty plaques within the arteries, is a top contributor to cardiovascular disease. Inflammation promotes atherosclerosis progression, increasing the likelihood of plaque rupture, which can lead to a stroke or heart attack. In the atherosclerotic environment, low-density lipoprotein (LDL) becomes chemically modified to form oxidized (oxLDL). Ingestion of these modified lipoproteins by macrophages promotes macrophage secretion of inflammatory cytokines. Complement protein C1q is typically known for its pro-inflammatory role as part of the classical complement signaling cascade. However, we have previously shown that C1q has a novel, anti-inflammatory role independent of the complement cascade: opsonization of modified LDL with C1q has the ability to polarize macrophages towards an anti-inflammatory (M2) phenotype, as opposed to the pro-inflammatory (M1) phenotype that is prevalent in the atherosclerotic environment. Differential transcriptomic analysis suggests that C1q may influence macrophage phenotype by modulating the levels of components of the NLRP3 inflammasome, a protein complex responsible for activation of inflammatory cytokines IL-1β and IL-18. We therefore hypothesized that C1q modulates macrophage polarization during clearance of modified lipoproteins by decreasing NLRP3 inflammasome activity. These studies were performed in primary human monocyte derived macrophages (HMDMs) and the macrophage-like THP-1 cell line. We treated macrophages with 10 µg protein/mL oxLDL, with or without 75 µg/mL C1q. Some cells were also treated with IFNg and LPS to promote M1 polarization. Cells and media were collected after three hours and twenty four hours incubation for analysis using quantitative RT-PCR and Luminex assay, respectively. Our data suggest that when bound to oxLDL, C1q downregulates IL-1β expression in both resting and M1-polarized macrophages. This suggests that this non-complement role of C1q may be important in dampening inflammation in the early atherosclerotic lesion. Discerning the molecular pathways that regulate inflammatory states of macrophages can contribute to the development of therapeutics for chronic inflammatory illnesses such as atherosclerosis. This research is supported by the National Institute of General Medical Sciences of the National Institute of Health under award number SC3GM111146.
Poster #: 90
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Metastasis, Breast Cancer, Stromal Cells
Project Title: Tumor-secreted factors elicit tumor-permissive behavior of mesenchymal stem cells and macrophages
Author List:
Meade, Kayla; Graduate, Biology, California State University, Northridge, Presenting Author
Kelber, Jonathan; Biology, California State University, Northridge
Abstract: Cancer metastasis – the process by which cancer cells are disbursed from the primary tumor site throughout the body and take up residence in new tissues – accounts for over 95% of cancer-related deaths. However, before cancer cells can survive in these “foreign” microenvironments, these new tissues must be primed and acquire tumor-permissive properties. While priming of the metastatic niche may begin during the earliest tumor-initiating events at the primary tumor site, very little is known about what factors are produced by the primary tumor cells that home to and prime different metastatic niche cell types. Understanding these events may lead to therapeutic interventions that cause various metastatic niches to lose their cancer-permissive character and prevent the growth of metastatic tumors. To address this unmet challenge, we employ in vitro methods using matched isogenic human and mouse breast cancer cell models that represent metastatic (4T1 and 10CA1a) or non-metastatic (67NR and 10CA1h) tumor types. We sought to evaluate how the factors that are produced from these two breast cancer cell groups affect two different cell types that are commonly found in the tumor microenvironment – macrophages (MACs) (RAW264.7) and mesenchymal stem cells (MSCs) (C3H10T1/2). We first tested the effect of extracellular matrix (ECM) proteins on the proliferation/survival, morphology and migration of RAW and C3H cells. We discovered that RAW cell proliferation/survival increases selectively in response to laminin, whereas collagen and fibronectin increase spreading and migration of a small population of these cells. We observed that 4T1 and CA1a cells acidify their growth media more readily than their non-metastatic counterparts. We also found that undiluted 4T1 and CA1a cell conditioned media significantly reduced MSC and MAC viability in comparison to the conditioned media from the non-metastatic breast cancer cells – an effect that was independent of ECM conditions. However, when diluted 1:1 with fresh media, 4T1 and CA1a cells stimulated C3H cell proliferation and RAW cell spreading, suggesting that the metastatic breast cancer cell secretome may increase the presence of tumor-promoting stromal cells. Future studies will evaluate the molecular profiles of these breast cancer cell-modified MSCs and MACs as well as differences in gene expression between metastatic and non-metastatic breast cancer cells that may account for this stromal cell changing behavior.
Poster #: 91
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Lipoprotein Lipase, Scavenger Receptors, cholesterol transporter
Project Title: Lipoprotein Lipase regulates the expression of genes responsible for cellular cholesterol uptake and efflux in human and mouse macrophages
Author List:
Kim, Aidan; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Moctar, Khadija; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Medh, Jheem; Chemistry and Biochemistry, California State University, Northridge
Abstract: Background: Cellular cholesterol accumulation is the net result of receptor-mediated cellular uptake and transporter-mediated efflux. Scavenger receptors (CD36, SR-AI, SR-BI) mediate the cellular uptake of cholesterol whereas the ATP-binding cassette transporter A1 (ABCA1) mediates the unidirectional efflux of excess cholesterol. SR-BI also mediates bidirectional cholesterol transport. Lipoprotein lipase (LPL) is secreted by macrophages and catalyzes the hydrolysis of circulating triglycerides. Macrophage LPL expression was shown to be inversely correlated with ABCA1 levels. We hypothesized that LPL may regulate the expression of macrophage scavenger receptors as well.
Objective: To examine if the expression of macrophage scavenger receptors and cholesterol transporter are regulated by LPL. Regulation of gene expression was studied in LPL-knock-down THP-1 macrophages and LPL-over-expressing mouse macrophages.
Methods: THP-1 monocytes were cultured and were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate. Thioglycolate-stimulated macrophages were isolated from the peritoneal cavity of transgenic mice with macrophage-specific LPL overexpression. The expression of CD36, SR-AI, SR-BI and ABCA1 was compared in normal and LPL- (THP-1) or LPL+ (mice) macrophages by RT-PCR analysis. b-actin served as a control for RNA mass. Gel images were quantified by ImageJ analysis.
Results: Silencing of the LPL gene in THP-1 macrophages correlated with a significant increase in ABCA1 expression (161% of WT). This was corroborated in mouse macrophages since overexpression of LPL resulted in a 50% decrease in ABCA1 expression. The expression of scavenger receptors SR-BI and CD36 was significantly lower in LPL- THP-1 cells compared to WT macrophages (55% and 60%, respectively, compared to WT). Contrary to results in THP-1 cells, LPL-overexpressing mouse macrophages showed a decrease in SR-BI levels (20% of WT), but no change in CD36 expression. The expression of SR-AI was not influenced by LPL silencing or overexpression.
Conclusion: Manipulation of LPL expression modulates the transcription of genes relevant to cholesterol accumulation in isolated THP-1 cells. In LPL+ transgenic mice, a decrease in SR-BI may be due to the influence of other organs in cholesterol homeostasis and SR-BI’s role in cholesterol efflux.
Acknowledgements: This work was supported by National Institutes of Health Awards R15HL083946 and SC3GM095413.
Poster #: 92
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Breast Cancer, Walnuts, Cyclin B1
Project Title: Identifying compounds from walnuts that target cell cycle proteins.
Author List:
Dsouza, Stephanie; Graduate, Biological Sciences, San José State University, Presenting Author
Garcia, Jasmine; Undergraduate, Biological Sciences, San José State University, Presenting Author
Peralta, Joebelle; Undergraduate, Biological Sciences, San José State University
White, Brandon; Biological Sciences, San José State University
Abstract: Background: Our lab is interested in the effects of walnuts on breast cancer cells. Previous results from our lab have shown that a walnut extract (WE) could kill MDA-MB-231 breast cancer cells via an apoptotic mechanism. We further showed that a WE could decrease Cyclin B1 mRNA and protein levels. Cyclin B1 proteins are elevated in breast cancer and hypothesized to be a potential therapeutic target for treatment. Therefore, our goal was to characterize isolated compounds from walnuts responsible for decreasing Cyclin B1 levels and to identify the mechanism by which Cyclin B1 decreased.
Methods: MDA-MB-231 cells were treated with DMSO, WE (0.25mg/ml), or 100 micromolar of the following compounds from walnuts: tellimagrandin I, casuarictin, gallic acid, pendunculagin, ellagic acid, or praecoxin A methyl ester for 24, 48 or 72 hours. For quantitative PCR, total RNA was isolated using TRIzol reagent. One microgram of total RNA was converted to cDNA using a SuperScript III Kit. cDNA was diluted 1:100 and used in a qPCR reaction with Power SYBR Green PCR master mix. qPCR was conducted on 7300 ABI qPCR system. For western blots, protein extracts were prepared in RIPA lysis buffer, quantitated by BCA assay, and 35-50ug of protein were ran into 10% SDS PAGE gels and electrophoretically transferred to PVDF membrane. The following antibodies were used for western blots: Cyclin B1, SP1, GAPDH, Cyclin E2, Cyclin H, and PARP-1.
Results: A significant decrease in Cyclin B1 mRNA levels was detected 24 hours after treatment with WE, tellimagrandin I and casuarictin. Other compounds tested did not affect Cyclin B1 mRNA levels. We also saw a decrease in Cyclin B1 protein levels when treated with tellimagrandin I. Cyclin B1 is reported to be regulated by SP1 transcription factor. We observed that SP1 protein levels were unaffected while qPCR data showed a decrease in Sp1 mRNA levels. We finally tested if Sp1 is prevented from binding to its DNA element and show that WE, tellimagrandin I, casuarictin and ellagic acid prevent SP1 binding in a dose dependent fashion.
Conclusion: Our results suggest that the mechanism of action of a WE ability to target breast cancer cells is through decreases in Cyclin B1thereby blocking cell cycle. The decreases in Cyclin B1 appear to occur through by the compounds preventing SP1 from binding to its DNA element in the Cyclin B1 promoter. Further analysis is continuing to look at other cell cycle proteins that are regulated by SP1.
Poster #: 93
Campus: CSU San Bernardino
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Influenza , Nucleoprotein , RNA helicase
Project Title: Role of N-terminus of Influenza Nucleoprotein in viral gene expression
Author List:
Lin, Jared; Graduate, Biology, California State University, San Bernardino, Presenting Author
Newcomb, Laura; Biology, California State University, San Bernardino
Abstract: Influenza is a pressing health issue yet many of the molecular mechanisms regulating virus propagation remain unclear. Our aim is to define biochemical interactions involved in viral RNA expression in order to identify antiviral targets and contribute toward the goal of developing innovative treatments. The influenza viral ribonucleoprotein complexes (vRNPs) are responsible for viral RNA synthesis. Each vRNP is comprised of one vRNA segment, the viral RNA dependent RNA polymerase (RdRP), and multiple copies of nucleoprotein (NP). NP serves as scaffold in formation of vRNPs, but also interacts with both viral and host factors, which likely regulates vRNP formation and activity. The N-terminus of NP contains a nonconventional nuclear localization signal (NLS1) essential for initial vRNP nuclear import, but which also interacts with host RNA helicases UAP56 and URH49 to enhance viral RNA replication in the nucleus. To examine the role of the N-terminus of NP aside from its nuclear localization activity, we constructed N-terminal 20 amino acid deletion mutants with or without the addition of the conventional NLS from SV40 T-antigen, termed del20NLS-NP and del20-NP, respectively. Previous work in our laboratory characterized del20NLS-NP and reported decreased viral gene expression exacerbated as vRNA template is lengthened, consistent with a lack of interaction with host RNA helicases. Here we show del20-NP maintains the defect in viral gene expression similar to del20NLS-NP in the context of reconstituted vRNPs, observed by fluorescent microscopy and western blot analysis of vRNA encoded proteins. Interestingly, our results reveal addition of the SV40-NLS at the N-terminus of NP hinders gene expression, as del20NLS-NP maintains a more severe defect in reconstituted vRNP function than del20-NP. This is consistent with our earlier observation that an N-terminal FLAG epitope tag fused to NP results in non-functional vRNPs. These data suggest perturbations of the N-terminus disrupt vRNP activity and support the N-terminal region of NP as a flexible interaction domain important not only in vRNP nuclear localization, but also efficient viral gene expression. Based on these observations, we expect addition of SV40-NLS to the N-terminus of wild type NP will inhibit viral gene expression. Once the N-terminus of NP is better defined and essential molecular interactions mapped in detail, it may be appreciated as a fruitful antiviral target.
Poster #: 94
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Cloning, manganese, transcriptional regulator
Project Title: Cloning of DNA binding proteins that may regulate expression of the manganese-oxidizing protein in Erythrobacter sp. SD-21
Author List:
Oseguera, Natalie; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Peterson, Justin; Undergraduate, Biological Science, California State University, Fullerton
Mao, Lucy; Undergraduate, Biological Science, California State University, Fullerton
Kim, SeongEun; Graduate, Biological Science, California State University, Fullerton
Johnson, Hope; Biological Science, California State University, Fullerton
Abstract: Transition metals can be toxic at high levels. Tactics to remove and control the abundance of toxins in the environment can be expensive. An alternative solution is bioremediation, which uses microbes to adsorb or modify pollutants. Manganese (Mn)-oxidizing bacteria can convert soluble Mn (II) to insoluble Mn (III, IV) oxides reducing its toxicity. In addition, Mn oxides are also able to adsorb metals such as mercury and arsenic. Although Mn oxidizing bacteria can be beneficial for bioremediation, it is unknown how these bacteria and their proteins are regulated and influence the fate of Mn. A manganese-oxidizing protein (MopA, encoded by mopA) was found in marine bacterium Erythrobacter sp. SD-21, but the regulation of this protein is unknown. The aim for this project is to determine the DNA binding regulatory proteins that control gene expression of mopA. The mopB gene is also expressed with mopA and encodes a potential DNA binding regulator. MopB may control the expression of the mopBA operon. Upstream of both genes, there are putative binding sites for the transcriptional regulator, OxyR, a transcriptional regulator that responds to hydrogen peroxide. Two putative oxyR genes are present in Erythrobacter sp. SD21. These putative DNA binding regulators and oxyR from Pseudomonas aeruginosa have been cloned into the pSpeedET expression vector and transformed into E.coli HK100 using the Polymerase Incomplete Primer Extension (PIPE) cloning technique. Colony PCR was performed to verify successful cloning. Plasmid DNA was purified and sequenced for confirmation of positive clones. MopB was expressed and purified by Nickel Affinity Chromatography. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to assess the purity. The expression of the OxyR proteins was very low and needs to be optimized. After proteins are expressed and purified, an electrophoretic mobility shift assay (EMSA) will be conducted to determine if they bind upstream of mopBA and regulate Mn oxidation. Acknowledgements: CSUF Biological Science Department, CSUPERB
Poster #: 95
Campus: CSU Long Beach
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Cerebral cortex, Sexual differentiation, Androgen receptor
Project Title: Androgen Regulation of Sexually Dimorphic Calbindin Expression in the Developing Mouse Cerebral Cortex
Author List:
Ramos, Layla; Undergraduate, Department of Biological Sciences, California State University, Long Beach, Presenting Author
Henson, Regina; Undergraduate, Department of Biological Sciences, California State University, Long Beach
Tsai, Houng-Wei; Department of Biological Sciences, California State University, Long Beach
Abstract: Calbindin-D28K, a calcium binding protein, is a biomarker for sexually dimorphic neuronal populations in the rodent brain. More calbindin-expressing neurons are present in the preoptic area and the bed nucleus of the stria terminalis of male mice than in females. Besides being intimately connected with the control of male sexual behavior, masculinization of calbindin expression in these two brain regions has been shown to be regulated by perinatal exposure to testosterone (T) in part via activation of androgen receptor (AR). Other than the hypothalamus, AR is also highly expressed in the cerebral cortex, which plays an important role in complex cognitive and social behaviors. Many of cortical functions are sexually dimorphic, but the neural mechanism underlying these differences between the sexes has not been well characterized. A recent study reported that sex differences in social and anxiety-like behaviors as well as gene expression in the prefrontal cortex and amygdala were eliminated in calbindin-knockout mice, suggesting that sexual dimorphism in calbindin expression and function might be present in non-reproductive brain regions, such as the cortex, and similarly regulated by AR as well. To test this hypothesis, we used immunohistochemistry to detect and analyze the distribution and number of calbindin-immunoreactive (ir) neurons in the forebrains of testicular feminized (Tfm) mice, lacking functional AR, and their wild-type littermates three weeks after birth. We first observed that calbindin-containing cells were widely distributed in the mouse cerebral cortex with labeled neurons mainly in layers 2/3 and 5/6, but very few in layer 4. Next, we counted the number of calbindin-expressing cells in the primary motor cortex (M1) and the forelimb region of the primary somatosensory cortex (S1FL). We found more calbindin-ir neurons in the M1 (318.0 ± 18.0) and S1FL (260.5 ± 8.4) of wild-type female mice than males (263.7 ± 18.2 and 162.5 ± 19.2) (p<0.05). Interestingly, Tfm males show female-biased upregulation of calbindin expression in the S1FL (252.5 ± 11.7), but not M1 (311.9 ± 13.4). Our preliminary data support a critical role for the AR in establishing partial sexual dimorphism in calbindin expression during early development as well as its potential links to non-reproductive neural function.
Poster #: 96
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: (p)ppGpp, polyhydroxybutyrate, starvation
Project Title: Three transcription factors regulate the accumulation of a dynamically-localizing polyester in Caulobacter crescentus
Author List:
Schreiner, Shannon; University of Southern California
Stott, Kristina; Jet Propulsion Laboratory
Vu, Son; Oliver View Medical Center
Bueno, Diana ; Pasadena City College, Presenting Author
Rocha, Alexa; Undergraduate, Biology, California State University, Northridge, Presenting Author
Arvizu, Ignacio; Undergraduate, Biology, California State University, Northridge
Murray, Sean; Biology, California State University, Northridge
Abstract: How organisms respond to starvation is poorly understood at the cellular level. This study focuses on the model organism Caulobacter crescentus, which evolved to survive long periods of starvation, to address this important question. To study this problem, we created a FabH-depletion strain to induce fatty acid starvation. In response to starvation various bacterial species produce the intracellular signaling molecule (p)ppGpp, which helps RNA polymerase turn on genes whose products promote survival under starvation conditions. We discovered that one set of (p)ppGpp-dependent genes leads to the production of the carbon storage molecule (and biodegradable plastic) polyhydroxybutyrate (PHB) in response to fatty acid starvation. Fluorescence microscopy, immunoblot, transcriptional reporters, and mutant strains were used to generate a model for PHB production in response to fatty acid starvation in Caulobacter crescentus. (p)ppGpp is responsible for the expression of the transcription factor CtrA, which activates the expression of the transcription factor PhaR, which is required for PHB accumulation. Thus, our model illustrates how three transcription factors work together to modulate expression of the PHB biosynthetic genes in response to fatty acid starvation.
This work was supported by NIH grant SC2 GM084860 and a CSUPERB Development grant to SM, NIH R25 GM063787 to MariaElena Zavala in support of IA, and CSUN Interdisciplinary Research Institute in the Sciences Fellowships to KS and SS.
Poster #: 97
Campus: CSU Long Beach
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: obesity, metabolism, development
Project Title: High Fat Diet-Induced Obesity Affects Development, Fertility, and Lifespan in Drosophila melanogaster
Author List:
Soto-Sauza, Karla; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Makary, Youstina H.; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Osman, Noor ; Graduate, Biological Sciences, California State University, Long Beach
Pieplow, Alice K.; Graduate, Biological Sciences, California State University, Long Beach
Eldon, Elizabeth D.; Biological Sciences, California State University, Long Beach
Abstract: According to the World Health Organization, obesity is a global epidemic. Drosophila melanogaster is an ideal model in which to study developmental and transcriptional responses to obesity because many mammalian lipid metabolic genes have orthologs in Drosophila. Here we investigate the correlation between excessive lipid uptake, triglyceride levels in the third larval instar, developmental timing, and lifespan. As expected, third instar larvae raised on a high fat diet (64.7% fat) accumulate significantly higher levels of triglycerides than those raised on control food (1.1% fat), as measured by triacylglycerol (TAG) assay (p=0.0111, p=0.0022). Expression of Krüppel (Kr), a transcription factor expressed in the fat body in response to secretion of the steroid hormone, 20-hydroxyecdysone, during early third larval instar, was analyzed by a lacZ fusion gene reporter. Optical density readings of the CPRG assay on third larval instars raised on normal and high fat food were taken every 0.5 hours for 3.5 hours. Assays at each time point indicate a strong significant difference in Kr expression levels (p<.0001). Transcript levels of both Kr and salivary gland secretion 3 (sgs3), a gene whose expression initiates during mid third larval instar, were also analyzed by quantitative polymerase chain reaction (qPCR). Preliminary data indicate that early third larval instars raised on a high fat diet have higher Krüppel expression than those raised on a normal diet. This may be due to the increased amount of triglycerides in their bodies, which may lead to increased steroid synthesis. SGS3 expression in early third larval instars is higher on a high fat diet. However, late third larval instars from both treatments express similar amounts of SGS3. Finally, counting progeny produced and adult survival revealed that lifespan and fertility are both reduced in flies raised on a high fat diet, though their progeny have normal lifespans when transferred to control food. We are continuing to analyze the fertility of multiple generations raised on control and a high fat diets as well as their survival under starvation stress. We plan to further understand how a high fat diet affects larval development by analyzing the expression of genes involved in lipid metabolism that have homologs expressed in mammalian tissues, as well as additional genes regulating development.
This project was funded by NIH grant through RISE program grant R25GM071638 and a CNSM small faculty grant.
Poster #: 98
Campus: San Francisco State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: microbe-host interactions, bacterial transcription, reporter enzyme assay
Project Title: Joint role of a protease and lipoprotein in symbiosis between rhizobia and legume plants
Author List:
Moore, Rebecca; Undergraduate, Biology, San Francisco State University, Presenting Author
Sapienza-Martinez, Joseph; Undergraduate, Biology, San Francisco State University, Presenting Author
Chen, Joseph; Biology, San Francisco State University
Abstract: Nitrogen is a fundamental element of life, but current agricultural practices quickly deplete its bioavailability from soil. While anthropogenic fertilizers can supply the nutrient rapidly, leguminous plants, such as alfalfa, offer a more environmentally sustainable alternative. These plants form a symbiotic relationship with rhizobial microorganisms which convert atmospheric nitrogen into ammonium, capable of being metabolized by the plant. Sinhorhizobium meliloti serves as a model bacterium for studying the cascade of molecular signals leading to this symbiosis. Exopolysaccharides produced by S. meliloti, in particular succinoglycan (or EPS-I), have been shown to be critical for successful infection of the plant root hairs. Two previously uncharacterized proteins, LppA (a lipoprotein) and JspA (a zinc-dependent protease), appear to regulate jointly EPS-I production, in turn affecting the efficiency of symbiosis. Microarray analysis identified over 100 S. meliloti genes whose expression changed when JspA was overexpressed. Based on such transcriptional changes, we hypothesize that LppA and JspA modulate the activity of a conserved signaling pathway integral to bacterial infection. Transcriptional fusions of the reporter enzyme beta-glucoronidase (GUS) to candidate downstream targets were constructed to validate the microarray results. Measurements of GUS activity under different conditions indicate that JspA and LppA indeed influence expression of genes that belong to the regulon of the conserved ExoR-ExoS-ChvI signaling pathway. Further investigation of the activities of LppA and JspA will help clarify the molecular events that allow S. meliloti to colonize its host plant effectively. Increased understanding of factors that contribute to rhizobia-legume symbiosis can help improve agricultural practices: finding methods to enhance symbiosis and nitrogen fixation may lessen reliance on fertilizers, thus reducing the ecological impact of human activities while improving crop yields.
This work was supported by the Arnold and Mabel Beckman Foundation, NSF, and NIH.
Poster #: 99
Campus: CSU Fresno
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Caenorhabditis briggsae, mitochondria, development
Project Title: Reactive oxygen species and developmental delay in Caenorhabditis briggsae
Author List:
Lauri, Marisol; Undergraduate, Biology, California State University, Fresno, Presenting Author
Ross, Joseph; Biology, California State University, Fresno
Abstract: To understand how species form, it is useful to study hybrid dysfunction. Hybrid dysfunction is evident when crosses between two individuals of the same species produce offspring that have reduced fitness (often sterility or lethality) relative to the parents. Such effects suggest that a combination of genes inherited from the parents causes poor health in the hybrid offspring. I study this process in the nematode Caenorhabditis briggsae. When two particular strains of C. briggsae are crossed, they produce a hybrid F1 generation worm. When these F1 hermaphrodites self-replicate, about 20% of the F2 generation exhibits developmental delay. Delayed individuals take about 33% longer to reach adulthood than their wild-type siblings. Developmental delay results from a genetic incompatibility. Detrimental genetic interactions in hybrids of other species are known to cause mitochondria to produce reduced amounts of adenosine triphosphate (ATP), and mutations in mitochondrial genes can cause retarded development. Mitochondrial biochemical dysfunction might connect the organismal phenotype of developmental delay to a genetic basis of hybrid delay: incompatible alleles involved in ATP production. My project seeks to identify the molecular/cellular basis for how developmental delay occurs, and I hypothesize that mitochondrial dysfunction reduces development rate. To test the hypothesis, I compare mitochondrial function in delayed F2 hybrids and their wild-type siblings by using a fluorescent dye to measure in vivo levels of reactive oxygen species (ROS). After conducting multiple experimental replications of this protocol, I have found that differences in ROS level do exist between delayed and wild-type hybrids: decreased levels of ROS correlate with developmental delay. This suggests the possibility that a hybrid genetic incompatibility interferes with oxidative phosphorylation, thus reducing ROS production (and presumably ATP production) and lengthening development time in those hybrids containing the dysfunctional allele combination. Future efforts will focus on conducting additional mitochondrial biochemical tests as well as exploring whether the same effects occur in other inter-population C. briggsae hybrids. This project was funded by Fresno State ASI and College of Science and Mathematics FSSRA grants.
Poster #: 100
Campus: CSU Dominguez Hills
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Zebrafish, Gene Expression, In Situ Hybridization
Project Title: Investigation of Gene Expression in Zebrafish Skin Using Bioinformatic Analyses and In Situ Hybridization
Author List:
De La Torre, Maricruz; Graduate, Biology, California State University, Dominguez Hills, Presenting Author, Eden Award Finalist
Yamamoto, Joselyn; Undergraduate, Biology, California State University, Dominguez Hills, Presenting Author
Sanchez, Steven; Undergraduate, Biology, California State University, Dominguez Hills
Cokus, Shawn; UCLA
Wang, Fang; Biology, California State University, Dominguez Hills
Abstract: Cell to cell interaction is vital for both the developmental processes and cell functions in multicellular organisms. At the 18 somite stage (SS) of zebrafish embryos the skin cells begin to interact with the peripheral axons of somatosensory (touch-sensing) neurons. The free endings of these peripheral axons grow and extend between the two epithelial layers (basal and periderm) that make up the skin of zebrafish embryos. In between 54 hours post fertilization (hpf) and 72 hpf the peripheral axons of somatosensory neurons become engulfed by the basal cells. Recent studies have suggested that a close interaction between somatosensory neurons and skin cells may not only be important for early development, but also for neuronal function and structure. Using whole transcriptome shotgun sequencing (RNA-Seq), we generated comprehensive gene expression profiles for the different skin cell layers and non-skin cells at three developmental stages (20 SS, 52 hpf, and 72 hpf) that correlate with the process of skin-neuron interaction. We used WEB-based GEne SeT AnaLysis Toolkit to conduct bioinformatic analyses on the RNA-Seq data and have discovered that the expression of various genes and cellular pathways are time and tissue specific. For example, aep1 and plek2 are two genes that are highly expressed in skin cells at specific developmental stages. Little has been known about these two genes we thus performed whole-mount in situ hybridization to further investigate the gene expression patterns of aep1 and plek2. Our current results have provided the first evidence that the aep1 gene is expressed in skin cells at the later developmental stages (52 hpf and 72 hpf) while no expression is detected at 20 SS using in situ hybridization. In addition, no expression in any other tissues was observed at these three developmental stages. Our continuous research in examining gene expression patterns will pave the way for future functional studies of genes that may be involved in cell-neuron interactions.
Funding provided by CSUPERB New investigator grant, CSUDH RSCA, and CSUDH Faculty Scholar Program
Poster #: 101
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: aminoglycoside, molecular docking, antibiotic resistance
Project Title: Gliding Through Resistance: Discovery of an Aminoglycoside 6′-N-Acetyltransferase type Ib [AAC(6′)-Ib] Inhibitor via Glide Molecular Docking
Author List:
Chiem, Kevin; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Jani, Saumya; Undergraduate, Biological Science, California State University, Fullerton
Fuentes, Brooke; Undergraduate, Biological Science, California State University, Fullerton
Lin, David; Graduate, Biological Science, California State University, Fullerton
Rasche, Maseline; Chemistry & Biochemistry, California State University, Fullerton
Tolmasky, Marcelo; Biological Science, California State University, Fullerton
Abstract: Aminoglycosides are potent antibiotics used in the treatment of life-threatening infections. Unfortunately, the relatively recent rise and spread of genes coding for aminoglycoside modifying enzymes resulted in an increase in resistant strains that undermines their effectiveness. One of the most prevalent aminoglycoside modifying enzymes is AAC(6’)-Ib, which catalyzes the inactivation of the antibiotic by promoting acetylation using acetyl-CoA as a donor substrate. Attempts to design new aminoglycosides that are resistant to the action of these enzymes have been unsatisfactory. We hypothesize that an alternative path to solve the problem could be the development of enzyme inhibitors. We search for small molecules by docking to the crystal structure of AAC(6’)-Ib using the Glide software. Enzymatic activity was determined using the Ellman’s reagent in vitro assay. Growth curves for in cellulo assays were conducted on microtiter plates with Mueller-Hinton broth at OD600 and monitored every 20 minutes. A total of 280,000 small molecules from the Chembridge library were analyzed using Glide, a molecular docking software for rapid protein-ligand binding affinities estimations and ligand rankings. The highest ranked compounds were evaluated using the in vitro assay, and those most active were tested as inhibitors in cellulo. A single compound, 1-[3-(2-aminoethyl)benzyl]-3-(piperidin-1-ylmethyl)pyrrolidin-3-ol (compound 1), was found to significantly reduce the acetylation of kanamycin A and amikacin by AAC(6’)-Ib with IC50 values of 39.7 and 34.9 µM, respectively. Moreover, when an amikacin-resistant clinical strain of Acinetobacter baumannii was challenged with a combination of amikacin and compound 1, bacterial growth was completely inhibited. We conclude that compound 1 is a strong candidate as a hit molecule for further optimization to identify one or more lead compounds.
This work was supported by Public Health Service grant 2R15AI047115-04 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Poster #: 102
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Robo, neural crest, migration
Project Title: Role of Slit/Robo signaling in Contact Inhibition Locomotion of neural crest cells.
Author List:
cooper, andre; Graduate, biology, California State University, Northridge, Presenting Author
nketiah, arnelle; Undergraduate, biology, California State University, Northridge, Presenting Author
reyes, michelle; Undergraduate, biology, California State University, Northridge
zuhdi, nora; Undergraduate, biology, California State University, Northridge
martinez, darwin; Undergraduate, biology, California State University, Northridge
giovannone, dion; Graduate, biology, California State University, Northridge
Abstract: Neural crest cells (NCC) emerge by delamination from the dorsal neural tube and give rise to various components of the peripheral nervous system in vertebrates. These cells change from a non-migratory to migratory state during epithelial to mesenchymal transition (EMT). NCC migrate to distant regions before further differentiating, but the mechanism controlling delamination and subsequent migration is not fully understood. Slit (chemorepulsive ligand) and its receptor Roundabout (Robo) have been shown to play a role in the migration of NCC in vertebrates. Slit and Robo proteins in NCC were observed to repel trunk neural crest cells. Thus Slit was limited in definition as a guidance cue for migrating neural crest cells but has been shown to stimulate motility while repulsing neural crest cells. Importantly, Slit has been classified to be a tumor suppressor gene. The present study looked further into the mechanisms by which Slit/Robo interactions affect neural crest cell migration. Previously, a microarray of Slit GOF revealed critical molecules in maintaining cell of non-motile, epithelial phenotype; many are known downstream of Slit/Robo signaling. Surprisingly, we also found new molecules previously not associated with Slit/Robo signaling. In addition, many of the genes regulated by Slit2 gain of function (GOF) either promoted or inhibited cell division and have been also identified in cancer cells. Our findings reveal a new Robo-dependent role of Slit2 in neural crest cells. Currently, our focus is directed towards Robo and its contact inhibition of locomotion (CIL) properties. The first step was to corroborate our microarray findings via RT-PCR and qPCR. From these experiments we found several genes that also play a role in CIL. Second, we are looking at live cells that express a mutated Robo that silences the signaling and then observing cell-cell interactions. Our preliminary results show that NCC depend on Robo signaling to migrate properly and affect their cell-cell interaction. Embryos electroporated with same mutated Robo showed also disrupted migration. These findings support a new role for Slit/Robo signaling in CIL.
Poster #: 103
Campus: San Diego State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Hepatitis C virus, Core protein, Cell signaling
Project Title: Preservation and nuclear transport of phospho-IkBalpha via the capsid protein of Hepatitis C virus
Author List:
Lopez, Rodrigo; Undergraduate, Biology, San Diego State University, Presenting Author
Skrypek, Katie; Undergraduate, Biology, San Diego State University, Presenting Author
Williams, Wesley; Graduate, Biology, San Diego State University
Wolkowicz, Roland; Biology, San Diego State University
Abstract: The Hepatitis C virus (HCV) primarily attacks the liver activating an aggressive immune response which can cause cirrhosis, intense inflammation and ultimately hepatocellular carcinoma known to be the fifth most common form of cancer. HCV belongs to the Flaviviridae family, and as such it is a positive single-strand RNA virus. Post-infection, the viral genome is translated into a single polyprotein thereafter processed into ten viral proteins, the first of these being the capsid or core protein. Although the core protein is primarily associated with the encapsidation of the viral genome and the formation of the viral particle, it has been proven to function also in conjunction with other proteins and display several subcellular locations post-infection.
Our previous research has hinted that post HCV infection, the phosphorylated form of the NFkappaB transcription factor inhibitor; Phospho-IkBalpha, is preserved from degradation and might be transported to the nucleus. In order to show the preservation of Phospho-IkBalpha via HCV core, we have analyzed the possible interaction between the viral core protein and Phospho-IkBalpha as well as their subcellular translocation. Through cellular fractionation and western blot techniques, we have proved nuclear localization of the viral core protein. Core nuclear translocation was further demonstrated by confocal microscopy. Moreover, using retroviral technology, we have established stable cell lines expressing non-mature and mature versions of the viral core protein in an inducible manner. We are now in the process of monitoring Phospho-IkBalpha in these cell lines, in the absence or presence of core, in time sensitive studies. We are also in the process of corroborating the interaction of core and Phospho-IkBalpha through immune-precipitation and biochemical tagging experiments. In order to test binding as well as to track sub-cellar localization we have used the ubiquitin degradation inhibitor bortezomib which forcefully aggregates cellular Phospho-IkBalpha.
In sum, this would be the first time that nuclear Phospho-IkBalpha is recognized in the presence of the HCV viral protein core. This in turn, will provide new understanding of the viral life cycle of the HCV and shed light into the role of Phospho-IkBalpha aggregation in the nucleus.
Poster #: 104
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: aging, stem cells, Drosophila
Project Title: Changes in genetic regulatory interactions during aging in the fruit fly Drosophila melanogaster
Author List:
Mazmanian, Aris; Undergraduate, Biology, California State University, Northridge, Presenting Author
Halajyan, Armen; Graduate, Biology, California State University, Northridge, Presenting Author
Loza-Coll, Mariano; Biology, California State University, Northridge
Abstract: With the noticeable increase in our life expectancies, efforts to ameliorate the deteriorating effects of aging and extend health span have become a new frontier for medicine and biotechnology. Recent work has brought attention to the effect that aging-induced decline in the activity of adult stem cells and tissue regeneration may have in several aging-associated pathologies. Our laboratory is specifically interested in how aging may cause alterations in gene expression within adult stem cells of the fruit fly Drosophila melanogaster.
The expression of most of our genes is regulated by several inputs that originate from complex regulatory networks, which detect, integrate and decode information about the overall state of a cell and its environment. Slight, random fluctuations in any one of those inputs are typically tolerated by the network, a phenomenon commonly known as “network robustness”. We are currently exploring the general hypothesis that genetic networks may lose robustness as cells age, making them more sensitive to internal and environmental perturbations and thus explaining the overall decline in stem cell function with aging. To begin exploring our hypothesis, we used genetically engineered flies that carry an activity reporter for the Signal Transducer and Activator of Transcription (STAT) pathway. We compared the response of the STAT pathway to a minor perturbation in stem cells of the testis or posterior midgut from young or aged flies. To perturb the network, we induced a brief down-regulation in the expression of Escargot (Esg), a transcription factor that is known to regulate stem cells in both tissues. Our results show that aging does not affect the response of the STAT pathway to a brief knockdown in Esg expression in stem cells of the midgut. In the testis, on the other hand, aging led to an intriguing reversal of the regulatory connection between Esg and STAT activity. In young males, a brief downregulation of Esg caused a noticeable decrease in STAT activity within hub cells, a cluster of cells that is critical for the maintenance of testis stem cells. In stark contrast, a similar inhibition of Esg in aged testes caused the opposite effect, i.e. an increase in STAT activity. These observations point to an aging-induced rewiring of the regulatory connections between Esg and the STAT in the testis, but not the midgut.
This work was supported by a CSUN start-up grant.
Poster #: 105
Campus: CSU San Marcos
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Arabidopsis thaliana, glutaredoxins, root growth
Project Title: Defining the expression domains of Arabidopsis thaliana glutaredoxin genes
Author List:
Davalos, Oscar; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Rosas, Miguel; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Fernandez, Francisco; Undergraduate, Biological Sciences, California State University San Marcos
Escobar, Matthew; Biological Sciences, California State University San Marcos
Abstract: Glutaredoxins are small redox enzymes that use glutathione as a substrate to reduce disulfide bonds in target proteins. Plants have far larger numbers of glutaredoxins than other organisms, largely due to a unique clade of class III glutaredoxins that is exclusively found in higher plants. Previously, we functionally analyzed a cluster of five class III glutaredoxin genes arranged in a tandem array on Arabidopsis thaliana chromosome 4, demonstrating that these genes act as negative regulators of primary root growth. The purpose of this study was to characterize the specific cell- and tissue-level gene expression domains of three of these glutaredoxin genes: AtGRXS5, AtGRXS6, and AtGRXS8. The promoter regions of each of these glutaredoxins were cloned upstream of the reporter gene GUS (beta glucuronidase) in a plant expression vector, which was used for plant transformation. Colorimetric GUS assays were then performed on the transgenic Arabidopsis plants expressing AtGRXS5pro::GUS, AtGRXS6pro::GUS, and AtGRXS8pro::GUS gene fusions. For AtGRXS6 and AtGRXS8, expression was localized exclusively in root and shoot vascular tissue, throughout plant development. AtGRXS5 displayed weak expression, exclusively in shoot vasculature. Expression of AtGRXS6 and AtGRXS8 was also significantly upregulated by nitrate, with GUS activity >6-fold higher in plants grown in media containing nitrate compared to nitrate-deficient media. Histological studies are currently underway to further define the cell-level localization of glutaredoxin gene expression within the vascular tissue. Collectively, our findings suggest that glutaredoxins may play an important role in nutrient signaling in plants, tying root system development to the availability of nitrogen in the soil. This research was supported by NIH SCORE grant SC3GM084721 to M.E.
Poster #: 106
Campus: CSU San Bernardino
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: C. elegans, neurobiology, calcium imaging
Project Title: Calcium imaging of a dopamine-regulated chemosensory circuit in Caenorhabditis elegans
Author List:
Kunkel, Cory; Graduate, San Bernardino, California State University, San Bernardino, Presenting Author
Kim, JunHyeong; , Biology, California State University, San Bernardino
Baidya, Melvin; Graduate, Biology, California State University, San Bernardino
Turturro, Tricia; Graduate, Biology, California State University, San Bernardino
Chao, Mike; Biology, California State University, San Bernardino
Abstract: The neurotransmitter dopamine regulates chemosensory avoidance behavior in the model organism Caenorhabditis elegans. Avoidance behaviors are mediated by the polymodal ASH nociceptive sensory neurons, and behavioral avoidance of stimuli detected by ASH is less robust when dopamine signaling is impaired. Our broad research goals are to understand the underlying molecular and cellular mechanisms of how dopamine modulates the activity of neural circuits. We hypothesize that dopamine plays a modulatory role on the ASH neurons, lessening the response of the ASH neuron to noxious environmental stimuli. To test our hypothesis, we are using a custom polymethylsiloxane (PDMS) microfluidics device and the transgenically-expressed Ca2+ sensor GFP variant G-CaMP to characterize the neural response of ASH neurons in wild type and dopamine-deficient cat-2 mutant C. elegans animals, which lack the dopamine biosynthetic enzyme tyrosine hydroxylase. Changes in Ca2+ levels in ASH neurons, which is an indicator of neural activity, is measured optically by video capture of G-CaMP fluorescence. Our preliminary data using the osmolyte glycerol to stimulate ASH neurons show that exogenous dopamine perfused into the recording buffer in the microfluidics device affects ASH neuron excitability in cat-2 mutants, consistent with our hypothesis. To further refine our research methods, we have tested a panel of soluble chemical stimulants in C. elegans behavioral assays to determine what chemosensory responses are modulated by dopamine. We have found that among the chemicals we tested, the response latency of cat-2 mutants to quinine was significantly decreased compared to wild type C. elegans strain, and the response of cat-2 animals to quinine was in the presence of exogenous dopamine, suggesting that dopamine plays a regulatory role in the chemo-sensation of quinine. Our ongoing efforts now focus on optimizing our assay conditions (e.g., recording buffer components) and identifying molecular elements in ASH neurons that mediate this response, including the relevant dopamine receptor(s).
Poster #: 107
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: SAX-3, Axon Termination, SLT-1
Project Title: Investigating a novel role for SAX-3/Robo and SLT-1/Slit in mediating axon outgrowth termination in C. elegans
Author List:
Coto Villa, Doris; Graduate, Biological Sciences, San José State University
Jimenez, Vanessa; Staff, Biological Sciences, San José State University
Thomas, Anthony; Undergraduate, Biological Sciences, San José State University
Kieu, Zanett; Graduate, Biological Sciences, San José State University
Zaroli, Johann; Graduate, Biological Sciences, San José State University
Duong, Thuy-Linh; Undergraduate, Biological Sciences, San José State University
Vargas, Christopher; Staff, Biological Sciences, San José State University
Chang, Eric; Undergraduate, Biological Sciences, San José State University, Presenting Author
Farooqi, Amber; Undergraduate, Biological Sciences, San José State University, Presenting Author
VanHoven, Miri; Biological Sciences, San José State University
Abstract: A critical step in the development of the nervous system is the outgrowth of axons, which are necessary for long-range transfer of information. However, little is known about the mechanism by which neurons terminate axon outgrowth. It is thought that molecular stop signals may instruct axons when to stop extending during development. Identifying these axon outgrowth termination signals and their receptors may be an important step understanding neurological disorders. With a simple and well-characterized nervous system, C. elegans is an ideal model organism in which to study axon outgrowth termination. In order to understand how axon outgrowth is mediated, we focus on two chemosensory neurons called PHBL and PHBR. Our genetic studies indicated that the transmembrane receptor SAX-3/Roundabout (Robo), previously isolated for its role in directing axons and cell bodies away from certain body regions, is also required for correct termination of axon outgrowth. PHB axons in sax-3/Robo mutant animals significantly overextend. Our goal is to investigate the role of SAX-3/Robo in axon outgrowth termination. We have determined that SAX-3/Robo functions cell-autonomously in PHB neurons. SAX-3/Robo is localized primarily to the distal region of the PHB axon and its cell body, and has lower levels at the tips of the dendrites. To determine if SAX-3/Robo functions during development to terminate axon outgrowth, or later to maintain axon length, we conducted temperature-shift experiments using a temperature-sensitive allele of SAX-3/Robo. Our results indicate that SAX-3/Robo functions during development to terminate axon outgrowth and is not necessary for maintaining axon length. Interestingly, the canonical SAX-3/Robo ligand SLT-1/Slit has an opposite phenotype to the overextension observed in sax-3/Robo mutants: PHB axons in slt-1/Slit mutants underextend. Our work indicates that axon outgrowth termination in our system is likely mediated by a conserved ligand-receptor pair. Our future goal is to elucidate the molecules that function downstream of SAX-3/Robo and SLT-1/Slit in axon outgrowth termination. This work is funded by the NIH (2SC3GM089595 to MV) and a CSUPERB Faculty-Student Collaborative Research Seed Grant to MV, and the following undergraduate fellowships: NIH MARC 5T34GM008253 to CV, VJ, and KB, and NSF REU DBI-1004350 to JZ.
Poster #: 108
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: CRISPR-Cas9, extracellular matrix, MAGP2
Project Title: Creating cell culture models to investigate the role of enzymatic cleavage on MAGP2 produced from the endogenous locus
Author List:
Song, Ann; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Price, Drew; Undergraduate, Chemistry and Biochemistry , California State University, Fullerton, Presenting Author
Perez, Aldwin Apollo; Graduate, Biological Science, California State University, Fullerton
Miyamoto, Alison; Biological Science, California State University, Fullerton
Abstract: Microfibril-associated glycoprotein 2 (MAGP2) is a small, secreted protein associated with elastic fibers in the extracellular matrix; it promotes elastic fiber formation, angiogenesis, anti-inflammatory effects, and regulates integrin and Notch signaling. Mutations that affect MAGP2 have been correlated with ovarian cancer and an inherited vascular disorder. One regulatory mechanism that modulates MAGP2 involves the cleavage of its last 20 amino acids by proprotein convertases (PC); this regulation was shown to promote the binding of MAGP2 to microfibrils via immunocytochemistry (ICC). However, regulation of matrix association by MAGP2 cleavage has only been tested at exogenous levels of MAGP2. Therefore, to study MAGP2 at endogenous levels in more biologically relevant conditions, cell culture models for both wild-type and mutant MAGP2 expression are being created using the CRISPR-Cas9 system. First, to induce endogenous wild-type MAGP2 expression, the CRISPR/ON system is being used. Preliminary RT-PCR results showed that one of six guide RNAs targeting the mouse MAGP2 promoter could active MAGP2 transcription when co-transfected with the nuclease-null Cas9 (dCas9) fused to a tripartite activator (VP64-p65-Rta), into mouse T3 ovarian tumor cells. Protein expression via Western blot and ICC are currently being performed. Second, in order to engineer loss-of-function MAGP2 mutations at its endogenous PC consensus site, the standard CRISPR-Cas9 technology is being used. Two guide RNAs have been identified that target the PC consensus site, and a donor plasmid has been generated that contains the PC site mutation to block cleavage of MAGP2. The guide RNAs, Cas9, and the donor plasmid have been co-transfected into normal human dermal fibroblasts that endogenously express MAGP2 and generate microfibrils (GM05659), and these cells are currently under the neomycin selection to create a stable GM05659-PC mutant cell line. These CRISPR-Cas9 modified cell lines will then be used in ICC assays to determine the role of cleavage on MAGP2 microfibril localization. By understanding MAGP2 in a more biologically relevant condition, MAGP2 can be better evaluated as an important therapeutic target. Funding: CSUF Junior Faculty Intramural grant, CIRM Bridges to Stem Cell Research.
Poster #: 109
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Leukemia, Blood, Cancer
Project Title: Growth receptor bound protein-2 (GRB2) overexpression increases proliferation of myeloid cells in acute myeloid leukemia (AML)
Author List:
Smith, Jesse; Graduate, Department of Biological Sciences, California State University, Chico, Presenting Author, Eden Award Finalist
Arpin, Carolynn; Chemistry, California State University, Chico
Stachura, David; Department of Biological Sciences, California State University, Chico
Abstract: Growth receptor bound protein-2 (GRB2) is an intracellular adapter protein responsible for linking receptor tyrosine kinase to downstream signaling proteins involved in cellular growth and differentiation. GRB2 overexpression has been identified in various forms of human cancer, but no research has investigated its role in acute myeloid leukemia (AML), a disease that causes an increase in myeloid cells, which are the body’s first defense against infection. AML is a relatively rare blood cancer that mostly affects older adults, but as the population ages, its rates are predicted to rise considerably. Importantly, AML is difficult to treat; there are multiple genetic and environmental components to the disease, and insensitivity to small molecule drug treatments after an initial period of remission is common. Importantly, many of the mutations involved in AML progression are in signal transduction pathways that are upstream of GRB2. For these reasons, we hypothesized that GRB2 overexpression would increase the proliferation of myeloid cells. Our findings demonstrate that GRB2 is overexpressed in the human myeloid leukemia cell line K562. Furthermore, we demonstrate that K562 cells stably overexpressing GRB2 in vitro show a nearly 2-fold increase in growth. Finally, we show that in vivo overexpression of GRB2 in zebrafish (Danio rerio) causes a 2-fold expansion of myeloid cells relative to mock-injected fish at 24 hpf. Our findings demonstrate a clear role for GRB2 overexpression in the proliferation of both normal and malignant myeloid cells. These finding highlight GRB2 overexpression as a potential biomarker and target for drug therapies to treat AML.
Poster #: 110
Campus: Humboldt State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Lgl1, Neural Progenitor Cells, mTOR
Project Title: A role for Lethal Giant Larvae 1 and mTOR in Differentiation and Anchorage Independent Growth of Murine Neural Progenitor Cells
Author List:
Bailey, Logan; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
Dombrowski, Matthew; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
Collins, Hannah; Undergraduate, Biological Sciences, Humboldt State University
Sprowles, Amy; Biological Sciences, Humboldt State University
Abstract: Lethal Giant Larvae (LGL) regulates cell polarity and the actin cytoskeleton. Lgl1 is tumor suppressor downregulated in glioblastoma, a deadly cancer of the central nervous system that can originate from mutations in neural progenitor cells (Klezovitch et al. 2004, Singh et al. 2004, Llaguno et al. 2009, Mangiola et al. 2007, and Gont et al. 2013). We wanted to investigate LGL1’s role in the differentiation and tumor forming potential of murine neural progenitor cells. Cells were isolated from the subventricular zone (SVZ) of two different adult Lgl loxP/loxP mouse brains and treated with AD-CRE GFP or AD-GFP to generate Lgl1 -/- and Lgl1+/+ cells lines, respectively. Cells were cultured as neurospheres and differentiated to the neuronal lineage or seeded in soft agar. Differentiation potential was assessed by morphology, expression of the astroglial marker glial fibrillary acidic protein (GFAP), and expression of the neural stem cell marker nestin. Tumor forming potential was analyzed by colony formation in soft agar. Our results show loss of LGL1 affects differentiated cell morphology but not nestin expression, GFAP expression, or soft agar colony formation. Cells exposed to chemicals known to regulate the JNK and mTOR signaling cascades did demonstrate genotypic specific differences. Lgl1 -/- cells treated with DMSO showed increased GFAP expression and colony formation in soft agar in when compared to Lgl1 +/+. Treatment with the JNK/mTOR activator anisomysin had a similar effect, but it was less robust. The mTOR inhibitor PP242 eliminated the Lgl1 dependent differences while the JNK inhibitor SP600125 was equally lethal to both cell lines. Our results suggest Lgl1 regulates the mTOR pathway to affect neuronal differentiation and anchorage independent growth in neural progenitor cells. Future studies will explore the additional downstream mechanisms responsible for the effects of DMSO alone.
Poster #: 111
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Apoptosis, Glucocorticoids, BIRC3
Project Title: BV6, an IAP antagonist, potentiates glucocorticoid-evoked apoptosis of CEM human leukemia cells
Author List:
Ortega, Carina; Undergraduate, Biology, California State University, Northridge, Presenting Author
Zhou, Shuo; Undergraduate, Biology, California State University, Northridge, Presenting Author
Medh, Rheem; Biology, California State University, Northridge
Abstract: Glucocorticoids (GCs) are effective therapeutic agents because of their capability to activate apoptosis in human acute lymphoblastic leukemia (ALL) cells. GCs trigger intrinsic and extrinsic apoptotic pathways through a gene regulatory effect on key pro- and anti-apoptotic genes that promote cytochrome c and Smac (Second Mitochondrial Activator of Caspase) mediated activation of caspases. Our laboratory is investigating molecular pathways that regulate apoptosis using a set of CCRF-CEM human derived ALL cell lines. CEMC7-14 and CEMC1-15 mE#3(ectopic expression of the transcriptional regulator E4BP4) are sensitive, and CEMC1-15 are resistant to GC-evoked apoptosis. In the sensitive cells, dexamethasone (Dex), a synthetic GC, upregulates proapoptotic genes, but paradoxically, also upregulates the anti-apoptotic gene BIRC3 (aka inhibitor of apoptosis protein, cIAP-2), which inhibits Smac and caspase activation. We are testing the hypothesis that Dex-mediated BIRC3 upregulation is a rescue mechanism of cells to escape apoptosis. The IAP antagonist BV6 is a Smac mimetic that can override IAP-mediated inhibition of apoptosis. We tested whether treatment with BV6 facilitated basal or Dex-mediated apoptosis. To investigate the potential of BV6 to affect cell viability, MTT assays were performed. BV6 alone evoked cell death on all three cell lines. Co-treatment of BV6 and Dex potentiated cell death by more than 50% in sensitive and resistant leukemia cells. To test the ability of BV6 to affect apoptosis, Annexin V labeling was visualized by epifluorescence microscopy. BV6 enhanced Annexin V labeling in control and Dex-treated cells, suggesting that inhibition of BIRC3 potentiated the apoptotic pathway. Our data support the hypothesis that Dex-mediated up-regulation of BIRC3 may be an anti-apoptotic rescue mechanism activated in parallel to the proapoptotic response in leukemia cells. The synergistic combination of GCs and Smac mimetics is relevant for the future of combined therapies for leukemia.
Acknowledgments: This research project was funded by the NIH (SC3 GM 081099) awarded to RDM and a BUILD-PODER scholarship to CO.
Poster #: 112
Campus: CSU Los Angeles
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: zebrafish, craniofacial, confocal
Project Title: Live tracking of pharyngeal arch development in wildtype and wdr68 mutant zebrafish
Author List:
Whitman, Taryn; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Zitser, David; Undergraduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Nissen, Robert; Biological Sciences, California State University, Los Angeles
Abstract: Birth defects are the leading cause of infant mortality in the United States and substantially contribute to long-term illness and disability. Three percent of all children born have a congenital defect, one third of whom have craniofacial abnormalities. Many congenital defects are due to disruptions of normal gene expression. Cranial Neural Crest Cells (CNCCs) are multipotent cells that originate from the neural tube and migrate into transient structures known as the pharyngeal arches (PA); they are reliant on intrinsic regulation and signaling from other cells to correctly migrate and differentiate. During development, the BMP and EDN1/EDNRA pathways converge to pattern the CNCCs of the first PA that give rise to the lower jaw (mandible). Auriculocondylar Syndrome (ACS) is a human congenital defect characterized by the malformation of the mandible and ears. ACS is caused by a loss-of-function mutation in the EDN1 gene that results in disruption of normal mandible patterning; the mandible instead resembles the upper jaw (maxilla). The sequence of the EDN1/EDNRA gene and its role in jaw development between human and zebrafish are highly conserved, making zebrafish an excellent model to study craniofacial morphogenesis. Wdr68/DCAF7 is a highly conserved scaffolding protein that is required for Edn1 expression. How wdr68 mediates Edn1 expression is still unclear, but it appears to interact with the BMP pathway upstream of Edn1. Null mutants for wdr68 (wdr68hi3812/hi3812) lack the ventral (Meckel’s) and dorsal (palatoquadrate) jaw cartilage structures. The Tg(sox10:mCherryCAAX) locus marks migratory and post-migratory CNCCs. Adult transgenic zebrafish carriers of the Tg(sox10:mCherryCAAX)/+; wdr68hi3812/+ loci were incrossed to produce zebrafish embryos harboring the sox10:mCherry reporter that were either wildtype at the wdr68 locus or wdr68hi3812/hi3812 mutants. Confocal microscopy was used to image the fluorescent CNCCs in wdr68 mutants and wildtype siblings from 17-somites (17 hpf) to prim-25 (36 hpf). A morphogenetic CNCC defect was present in wdr68 mutant embryos by 24 hpf, shortly after the normal onset of edn1 expression, in regions fated to be M and PQ. Using the same confocal assay, wdr68 mutants treated with a BMP agonist isoliquiritigenin (ISL) were imaged revealing a partial rescue of M and PQ regions in wdr68 null mutants. This data reveals that wdr68 does interact with the BMP pathway to target downstream effectors in jaw development.
Poster #: 113
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Shewanella, antibiotic resistance, genome analysis
Project Title: Mobile Genetic Elements and Antibiotic Resistant Determinants Present in the Uncommon Pathogen Shewanella decolorationis
Author List:
Lazzaro, Terese; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Uong, Sylvia; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Montaña, Sabrina
Ramirez, Maria Soledad; Biological Science, California State University, Fullerton
Abstract: Antibiotic resistance has become a prevalent issue in healthcare but as well as the scientific community. The lack of knowledge and excessive use of antibiotic has led to bacterial acquisition of antibiotic resistant determinants via horizontal genetic transfer. Pathogens that were once considered “safe” are now rendered untreatable.
The goal of this project is to investigate the molecular mechanisms that explain the multidrug resistant phenotype expressed by a clinically isolated strain of Shewanella decolorationis (Shew256) recovered from a patient. Preliminary results obtained from a minimal inhibitory concentration (MIC) test were performed and revealed high levels of antibiotic resistances to cefepime, ceftazidime, and ciprofloxacin. From here total DNA extractions were performed and polymerase chain reactions (PCR) were completed. In addition, complete genome sequencing of Shew256 was performed with Illumina MiSeq-I and Nextera XT DNA library was used for sample preparation. Genomic analysis was carried out to expose the antibiotic resistant determinants using different bioinformatics tools including: BLAST (version 2.0), ACT (Artemis), ARG-ANNOT, ISFinder, etc. Moreover, PCR reactions were performed.
Genomic analysis and PCR reactions revealed the presence of different resistance genes, including the presence of a blaPER-2, an extended β-lactamase that explains its resistance profile against cephalosporins. Moreover, class 1 integron containing the resistance genes aar3, dfrA27, and aadA16 was found. Furthermore, a transposon containing aph(3’) within IS26 was found. Other genes codifying for resistance against teracyclines, sulfonamide and β-lactams were found.
In conclusion, evidences of acquisition of resistance genes that explain the multidrug resistance phenotype were observed. This Shewanella decolorationis strain is a prime example of how dangerous antibiotic resistance can be and the need to continue this line of research.
Poster #: 114
Campus: San Diego State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Microbiology, Metagenomics, Biofilm
Project Title: Metagenomic analysis uncovers strong relationship between periodontal pathogens and vascular dysfunction in American Indian population
Author List:
Korategere Vijay Kumar, Prathik; Graduate, Bioinformatics, San Diego State University, Presenting Author
Gottlieb, Roberta A. ; Biology, San Diego State University
Calac, Dan
Delange, Nicole; Staff, Institute for Public Health, San Diego State University
Lindsay, Suzanne; Institute for Public Health, San Diego State University
Penn, Tanya E.; Institute for Public Health, San Diego State University
Kelley, Scott T.; Biology, San Diego State University
Abstract: Periodontal disease (PD) is a well known risk factor for cardiovascular disease (CVD) but the casual relationship, if any, is not understood. American Indians and Alaskan Natives (AIAN) have high rate of both PD and CVD and a better understanding of how PD might affect heart health would be particularly helpful in this population. In this study, we sequenced the bacterial biofilms of periodontal (gum) pockets and used metagenomic sequencing and vascular health measurements (immune cytokine profiles and vascular flow) to determine the relationship of microbial pathogens and CVD. Twelve subjects were sequenced before and after standard periodontal treatment (24 sample). Other measures taken before and after treatment included a full dental screening; blood samples obtained to determine serum concentration of C-Reactive Protein, Interlukin-6, Interlukin-10, Interlukin-1ß, Tumor Necrosis Factor-α (TNF), Interferon-ɣ and cTnI; Fasting venous blood obtained to determine lipid profiles, and plasma glucose concentrations. The non-invasive Laser Doppler Fluxmetry (LDF) procedure was conducted to measures the microvascular vasodilation. We found highly significant relationships between the total abundance of 4 periodontal pathogens, Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia and Treponema denticola, and the inflammatory cytokine IL-1β (p=0.009) as well as with vascular flow post sodium nitroprusside (SNP) treatment (p=0.006). Two bacterial species mostly correlated to IL-1β were Fusobacterium nucleatum and Porphyromonas gingivalis. IL-1β has been strongly implicated at a causal factor in atherosclerosis and in periodontal bone loss. To our knowledge, this is the first direct link between abundance of specific periodontal pathogen and cardiovascular disease in humans, and suggests that these pathogens could be used as indicators for cardiovascular health.
Poster #: 115
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: supercoiling, transription, cell cycle
Project Title: Expression of the Caulobacter crescentus sciP promoter is modulated by gyrase-mediated supercoiling
Author List:
Heasley, Keaton; Undergraduate, Biology, California State University, Northridge, Presenting Author
Kumar, Priya; Undergraduate, Biology, California State University, Northridge
Requena, Kiana; Undergraduate, Biology, California State University, Northridge, Presenting Author
Zamora, Michael; Undergraduate, Biology, California State University, Northridge
Murray, Sean; Biology, California State University, Northridge
Abstract: The location of genes on the Caulobacter crescentus chromosome has been hypothesized to drive gene expression based on the methylation state of promoters. However, as the replisome copies the chromosome, it also mediates major changes in the supercoiling of DNA. In this study, we hypothesize that the transcription of some cell-cycle regulated genes is controlled by DNA supercoiling. To explore this possibility, we treated Caulobacter crescentus cultures with the antibiotic Coumermycin A1, which inhibits the activity of DNA gyrase. Although high levels of the antibiotic inhibit growth, low levels actually improve growth, as reported for other bacteria. SDS-PAGE analysis of cell lysates suggests that most C. crescentus proteins do not significantly change in abundance during Coumermycin A1 treatment. However, several proteins exhibited significant changes in accumulation. Immunoblots confirmed that one of these proteins is the cell cycle master regulator SciP. Since no major changes in the accumulations of the other master regulators—DnaA, GcrA, CtrA, or CcrM—were observed by immunoblot, only a subset of genes appear to be regulated by DNA supercoiling. Miller Assays, performed on cultures with either low or high levels of Coumermycin A1, confirmed that the reduction in SciP accumulation resulted from a decrease in sciP transcription. As gyrase activity becomes inhibited, sciP transcription is reduced. We also confirmed that SciP accumulation is reduced in cells treated with the gyrase-inhibitor novobiocin. Thus, DNA supercoiling plays an important role in the transcription of at least one bacterial cell cycle master regulator in C. crescentus.
This work was supported by NIH R25 GM063787 to MariaElena Zavala in support of KH and by NIH 8TL4GM118977-02 to Crist Khachikian in support of PK and KR.
Poster #: 116
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: methylotrophy, iron, lanthanide
Project Title: Expression of lanthanide uptake genes in Methylobacterium extorquens is controlled by the iron uptake regulators, Irr and FecI
Author List:
Kanda, Ramen; Undergraduate, Biological Sciences, San José State University, Presenting Author
Wingett, Justin; Graduate, Biological Sciences, San José State University, Presenting Author
Doherty, Jennifer; Undergraduate, Chemistry, San José State University
Tamsir, Bryan; Undergraduate, Biological Sciences, San José State University
Saglimbeni, Marco; Undergraduate, Biological Sciences
Skovran, Elizabeth; Biological Sciences, San José State University
Abstract: Methylobacterium extorquens is a model organism for the study of methylotrophy and has recently been shown to have the unique ability to use rare-earth elements (lanthanides) for methanol oxidation. We are currently engineering M. extorquens as a platform for the recovery of these rare-earth elements from mining ores and discarded electronics. In order to increase the efficiency at which M. extorquens recovers these metals, the genes involved in lanthanide uptake and regulation needed to be identified. Transposon mutagenesis identified a cluster of genes predicted to be involved in lanthanide uptake and includes: a TonB dependent receptor, an ABC transporter and several exported genes. Growth studies confirmed that these genes are required for lanthanide-dependent methanol growth. The presence of a TonB-dependent receptor suggests that there may be overlap between the systems involved in lanthanide and iron uptake. Null mutations were constructed in putative regulators of iron uptake including irr, fecI and fecR. Transcriptional reporter fusions were created which fused the predicted promoter regions of various iron uptake genes and the lanthanide transport cluster upstream of a gene encoding the fluorescent reporter, Venus, and expression was measured as an output of fluorescence. Expression was measured in the presence and absence of iron and lanthanum. A number of genes predicted to be involved in iron uptake showed differential regulation in response to iron while some also showed increased expression in the presence of lanthanum. Excitingly, preliminary results suggest that expression of the predicted lanthanide uptake cluster is controlled by the iron regulators, Irr and FecI and is differentially regulated by exogenous lanthanides but not iron. Taken together, these results suggest there is cross regulation between iron and lanthanide uptake and that the transcriptional regulators involved in this process can distinguish between these two metals. This is the first work to identify and characterize genes required for lanthanide uptake. Funding for this project was provided by San José State University.
Poster #: 117
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: whooping cough, vaccination, mesenchymal stem cells, ,
Project Title: mBM-Mesenchymal Stem Cells as a vehicle for immunization of mice against Bordetella pertussis
Author List:
Arjun, Sreelakshmi ; Graduate, Biology, San José State University, Presenting Author
Touhidul, Sohan; Undergraduate, biology, San José State University
Abramson, Tzvia; Biology, San José State University
Abstract: Bordetella pertussis (Bp) is an extracellular bacterial pathogen that causes pertussis whooping cough. While not fatal to adults, whooping cough is much more dangerous to infants resulting in approximately 100,000 deaths in per year worldwide. Current pertussis acellular vaccine available is not effective in providing lifetime immunity to the disease and needs an additional booster after a period of 10 years.
Mesenchymal Stem Cells (MSC) derived from bone marrow is well documented to lack immunogenic features and demonstrate anti-inflammatory characteristics. Additionally, in vitro treatment of MSCs with IFNγ was previously shown to increase expression of antigen-presenting molecules such as MHC class II. Therefore in this study, we hypothesis that MSC can serve as an immunization vehicle to deliver B. pertussis antigens.
We used flow cytometry and immunofluorescence to demonstrate that MSCs isolated from C57BL/6 mouse bone marrow, express high level of MHC II in the presence of IFNγ. MSC were then pulsed overnight with heat-killed Bp and intranasally administered (one million/mouse) twice with one-week interval. Mice were then challenged with Bp infection. Effectiveness of the immunization was verified by checking lung bacterial load, and WBC responses at 5 dpi. The preliminary results demonstrate that mice that were immunized with MSC/Bp were 100% protected from the infection challenge as measured by the bacterial load in lungs at 5dpi. Flow cytometric analysis revealed more circulating B cells and monocytes in MSC/Bp immunized mice compared to control mice. Survival assay further proved that all the mice immunized with MSC/Bp survived high dose of Bp infection, whereas two out of three mice in the control group died.
These preliminary data suggest that BM-MSCs can serve as a vector to protect mice from Bp infection. We will further investigate for the precise immune responses induced by MSC/Bp vaccine.
Poster #: 118
Campus: San Diego State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila melanogaster, Gene Expression, Transcription Factors
Project Title: Assembly of SNAPc and TFIIIB on a Drosophila U6 snRNA gene promoter
Author List:
Verma, Neha; Graduate, Biology, San Diego State University, Presenting Author
Moreno, Ann Marie; Graduate, Biology, San Diego State University, Presenting Author
Kang, Jin Joo; Postdoc, Chemistry and Biochemistry, San Diego State University
Stumph, William; Chemistry and Biochemistry, San Diego State University
Abstract: In higher eukaryotes, RNA polymerase III (Pol III) promoters at U6 snRNA genes consist of a TATA box, recognized by TFIIIB and a proximal sequence element, PSE, recognized by the small nuclear RNA activating protein complex (SNAPc). In the fruit fly Drosophila melanogaster, DmSNAPc consists of three subunits DmSNAP190, DmSNAP50, and DmSNAP43; likewise TFIIIB also consists of three subunits, most commonly TBP, Brf1 and Bdp1. At Drosophila tRNA and 5S RNA promoters, TBP-related factor 1 (TRF1) is utilized in place of TBP, whereas at U6 promoters the canonical TBP is utilized for Pol III transcription (Verma et al. 2013, JBC 288, 27564-27570).
Site-specific protein-DNA photo-cross-linking studies of DmSNAPc and TFIIIB to U6 promoter DNA indicate that Bdp1 is in close proximity to DmSNAP43 and DmSNAP190 on the U6 promoter. This suggests that the interaction between DmSNAPc and TFIIIB may be mediated, at least in part, by Bdp1. We have investigated this further by electrophoretic mobility shift assays (EMSAs). Surprisingly, we have found that DmSNAPc, when bound to the U6 PSE, can recruit Bdp1 to the DNA in the absence of TBP and Brf1. Furthermore, EMSAs indicate that the DmSNAPc-Bdp1 complex, when bound to U6 promoter DNA, can recruit TBP to form a DmSNAPc-Bdp1-TBP-DNA complex. To investigate whether the TATA box is required for Bdp1 and TBP recruitment, the U6 TATA box was mutated to an unrelated sequence. Although mutation of the TATA box interfered with the recruitment of TBP, it did not prevent the recruitment of Bdp1 by DmSNAPc.
Additionally, a body of previous work from our lab has shown that DmSNAPc binds to U6 and U1 promoters (transcribed by RNA polymerase II) in distinct conformations. Interestingly, when we switched the U6 PSEA to a U1 PSEA by a 5-nucleotide change, DmSNAPc was unable to recruit Bdp1. This suggests that a surface of DmSNAPc that interacts with Bdp1 may be occluded when DmSNAPc binds to a U1 PSEA. Various DmSNAPc truncation and alanine scanning mutants are being utilized to investigate the specific region of DmSNAPc responsible for the recruitment of Bdp1 to the U6 promoter (Supported by the National Science Foundation and in part by the California Metabolic Research Foundation.)
Poster #: 119
Campus: CSU Fresno
Poster Category: Other
Keywords: prostate cancer, curcumin, medicinal chemistry
Project Title: Structure-Activity Relationship Studies of 1,5-Bis(1-Substituted-1H-Imidazol-2-yl)- penta-1,4-diene-3-ones in Prostate Cancer Cell-Models
Author List:
Patanapongpibul, Manee; Graduate, Department of Chemistry, California State University, Fresno, Presenting Author
Zhang, Xiaojie; Graduate, Department of Chemistry, California State University, Fresno
Chen, Guanglin; Staff, Department of Chemistry, California State University, Fresno
Peres, German Guiz; Undergraduate, Department of Chemistry, California State University, Fresno, Presenting Author
Chen, Qiao-Hong; Department of Chemistry, California State University, Fresno
Abstract: Curcumin is the major chemical component of Curcumin longa that has long been used as food seasoning and traditional medicine. Its anti-prostate cancer potential has been proven by in vitro cell-based experiments and in-vivo animal studies. However, its moderate potency and poor bioavailability make it as a lead compound rather than a FDA-approved therapeutics. Our research group has previously identified 1,5-bis(1-alkyl-1H-Imidazol-2-yl)penta-1,4-diene-3-ones as a group of promising curcumin analogs possessing up to 200-fold greater potency than curcumin in prostate cancer cell models. However, one optimal analog only exhibits 2-10 folds increase in mouse plasma concentration.
To pave the avenue for optimization of this group of curcumin analogs as chemotherapeutics for the treatment of prostate cancer, the present research aims to systematically explore their structure-activity relationships on prostate cancer cell models. To achieve this aim, over ten new analogs have been synthesized to investigate the effects of the N1 substituent group of the imidazole rings and of the double bonds of the central linker on anti-proliferative potency. The Horner-Wadsworth-Emmons reaction was employed as the key step reaction for the synthesis of these curcumin analogs. The NMR data were used to characterize their chemical structures and the WST-1 cell proliferation assay was used to assess their potency in three prostate cancer cell lines. Our data showed that i) 1,5-bis(1H-imidazol-2-yl)penta-1,4-diene-3-one, the potential metabolic product of 1,5-bis(1-alkyl-1H-imidazol-2-yl)penta-1,4-diene-3-ones, is much less potent than its parent compound; ii) introduction of a bulky group to the N1 position of the imidazole rings reduces the potency; and iii) reduction of the double bonds of the central linker is detrimental to the potency. We can conclude from these data that i) it is imperative to further improve the bioavailability of 1,5-bis(1-alkyl-1H-imidazol-2-yl)penta-1,4-diene-3-ones by introducing an appropriate metabolic stable group to N1 position because its potential metabolic product is inactive; ii) the double bonds in the central linker are necessary for the potency; and iii) an appropriate size of the N1 substituent of imidazole rings is important for the potency.
We are grateful to (i) CSUPERB for 2013 New Investigator and 2015 Research Development Awards, and (ii) CSU-Fresno for an Undergraduate Research Grant (to G.R. Peres).
Poster #: 120
Campus: CSU Los Angeles
Poster Category: Other
Keywords: Pseudomonas aeruginosa, Innate immunity, Biofilm
Project Title: Investigating the Mechanism of Activity of Antimicrobial Lipids Formulated in Liposomes
Author List:
Bush, Mabel; Undergraduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Ybarra, Tiffany; Undergraduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Fujii, Gary; Molecular Express, Inc.
Chiang, Su-Ming; Molecular Express, Inc.
Porter, Edith; Biological Sciences, California State University, Los Angeles
Abstract: The innate immune system provides the first line of defense against invading pathogens. Antimicrobial lipids including cholesteryl esters (CEs) have been increasingly recognized to play an important role as effector molecules. Preliminary data suggested that, when formulated in liposomes, cholesteryl arachidonate (CA) and cholesteryl linoleate (CL) inhibit bacterial biofilm formation. Understanding the mode of action of antimicrobial lipids may lead to novel approaches to combat multidrug resistant microorganisms. Among the leading causes of health care associated infections with multidrug resistant opportunistic bacteria are Pseudomonas aeruginosa (PA), a Gram-negative rod, and Staphylococcus epidermidis (SE), a Gram-positive coccus. We seek to understand how antimicrobial lipids act against bacteria in the hope of developing new drugs. Here, we wished to assess whether CA and CL containing liposomes fuse with bacteria using a fluorescence tandem approach. Bacteria were labeled with the live green fluorescent probe BCECF. Liposomes (Lip) containing CA (CA-Lip) or CL (CL-Lip) or liposomes without CEs (Lip) were loaded with the red fluorescent Texas Red Dextran. Bacteria and liposomes were incubated for up to 19 h and red fluorescence was measured hourly using an excitation wavelength that maximally excited BCECF but Texas Red only to a small degree. Upon fusion the BCECF emission wavelength would strongly excite Texas Red generating a pronounced increase in red fluorescence. In addition, biofilm was quantified at the end of the incubation. In 2 independent experiments conducted in triplicate, we found that biofilm production was reduced in PA and SE to < 50% of the control by CA-Lip and CL-Lip and by Lip to 65-70% of the control. Unexpectedly, a decline of red fluorescence after 10 h incubation was observed when liposomes were incubated with PA but not with SE or buffer only. This decrease was most prominent for CA-Lip (reduction to 44 ± 4.8 % after 19 h for PA compared to 85 ± 1.4 % for SE and 89 ± 10.3 % for buffer only). This data indicates that the mechanism of action differs between the Gram-negative PA and Gram-positive SE. Furthermore, while biofilm is inhibited in PA, planktonic bacteria may degrade the liposomes, possibly through secretion of lipases. Future drug development needs to take into account growth phase-dependent bacterial responses. Acknowledgements: CSULA Seed Grant, NIH R25 GM061331, 1SC1GM096916.
Poster #: 121
Campus: San Diego State University
Poster Category: Other
Keywords: Coral reefs, bacterial virulence, metatranscriptomics
Project Title: A metatranscriptomic approach for characterizing host immune responses to bacterial virulence factors in the coral holobiont
Author List:
Macherla, Saichetana; Graduate, Bioinformatics, San Diego State University, Presenting Author
Robinett, Nathan; Postdoc, Biology, San Diego State University
Little, Mark; Graduate, Biology, San Diego State University
Dinsdale, Elizabeth; Biology, San Diego State University
Wegley Kelley, Linda ; Biology, San Diego State University
Rohwer, Forest; Biology, San Diego State University
Abstract: Coral reefs are hotspots for biological diversity harboring some of the highest metazoan biomass in the sea. Corals live in symbiosis with photosynthetic dinoflagellates (zooxanthellae) and many other microscopic organisms collectively referred to as the coral holobiont. Studies have shown that there is significant exchange of dissolved substrate across the interface where corals and algae interact, which promotes high microbial abundances and activity. Since it has been shown that microbial communities associated with the coral-algal interface are enriched with members closely related to known bacterial pathogens, we hypothesized that the virulence factors (VF) transcribed by these microbes will trigger an immune response by the coral animal such as activation of the tumor necrosis factor (TNF) ligand, which initiates the pathway that leads to cell apoptosis. To our knowledge, no studies have shown an association between virulence factor proteins and host immune response at the coral-algal interfaces. Tissue punches across coral and algal interface were collected from Southern Line Island atolls. Samples were frozen in RNA later for transport back to the lab, where nucleic acids were extracted; libraries were then constructed and sequenced using Illumina technology. Ten clean RNA and DNA sequence libraries from two islands which represent five punches across the coral-algae or heterospecific coral-coral interactions, were included in the study. Both host immune responses (including the TNF pathway) and the relative abundances of virulence factor transcripts were estimated through sequence similarity comparisons to existing databases using BlastX. These preliminary comparisons demonstrate an up-regulation of TNF ligand at both coral-coral and coral-algae interfaces. Further studies, which will include more metatranscriptomes are required to determine which VF gene activates the TNF ligand at both coral-coral and coral-algal interactions.
Poster #: 122
Campus: CSU Long Beach
Poster Category: Other
Keywords: Drug-Eluting Stent, Metal Organic Framework, Thin Film
Project Title: Using Metal Organic Framework Film as a Drug-Eluting Stent Coating
Author List:
Sua, Andy; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Carachure, Lester; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Tian, Fanguyan; Chemistry and Biochemistry, California State University, Long Beach
Abstract: A significant proportion of patients are under high risk of thrombosis caused by polymer-based drug eluting stent (DES) implantations. We aim to overcome this problem through the development of biodegradable metal organic framework thin films that are suitable for chemical binding on metal stents to release drugs at a steady rate. Ultimately, we hope this type of inorganic thin film coating will shift the paradigm of current DES drug delivery systems, and will benefit those who suffer from vascular diseases and need a stent. In this study, MIL-88B (MIL stands for Material from Institut Lavoisier) was synthesized through hydrothermal method at 100oC. This novel porous inorganic framework is composed of iron-trimers connected with 1,4-benzendicarboxylic acid. Our X-ray diffraction and infrared spectroscopic studies confirmed its crystalline structure and chemical compositions. The drug uptake and elution of ibuprofen with MIL-88B was investigated by UV-Vis spectrometer combined with surface plasmon resonance (SPR) spectroscopy. After exposure to 1 uM ibuprofen in hexane for 30 min at room temperature, MIL-88B solution exhibited the lowest UV intensity of ibuprofen indicating the drug loading saturation point. The drug elution study was performed by incubating ibuprofen loaded MIL-88B in a standard phosphate-buffer saline (PBS) solution. A gradient increase in UV absorbance indicated a controlled drug release rate over the time frame of 120 min at room temperature. The SPR results show 1.2 degrees refractive angle change after 10 cycles of the layer-by-layer deposition of MIL-88B on an alkane thiol self-assembled monolayer terminated gold substrate. This helps us to understand the mechanism of MIL-88B film formation on a substrate: iron-containing clusters were formed before framework buildup, which was also confirmed by our microscopic studies. This study can be further applied to different classes of drugs with a variety of applications such as antiplatelets and immunosuppressants. The authors would like to acknowledge CSUPERB for the support of this project.
Poster #: 123
Campus: CSU Northridge
Poster Category: Other
Keywords: Drosophila, gametes, drug tolerance
Project Title: Repeated exposure of fruit flies to intoxicating doses of ethanol alters the initial response of their progeny to the drug
Author List:
Mcpherson, Michael; Undergraduate, Biology, California State University, Northridge, Presenting Author
Bonilla, Michelle; Undergraduate, Biology, California State University, Northridge, Presenting Author
Loza-Coll, Mariano; Biology, California State University, Northridge
Abstract: In recent years, there has been increased interest in the inheritance of enhanced metabolic responses to environmental conditions, such as diet, drug tolerance and addiction. For instance, the progeny of females exposed to a high-fat diet have a significantly higher propensity than control populations to developing metabolic syndrome and cardiovascular disease when exposed to a fatty diet. Despite the epidemiological evidence supporting this and similar observations, their genetic and molecular underpinnings remain largely unknown. While the available evidence points to epigenetic modifications of DNA (chromatin remodeling and DNA methylation) as two major underlying mechanisms of such inherited propensities, the source of epigenetic imprinting is less clear. Moreover, the fact that mammalian fetuses develop within a womb represents a significant challenge to identifying the original source(s) of such modifications. It is difficult to separate the role of regulatory imprinting of gametes produced in parents living under a specific environmental condition from epigenetic modifications that occur directly in a developing embryo, when the embryo is exposed to the same or very similar environments during gestation.
To circumvent this problem, we use the fruit fly Drosophila melanogaster, an insect with external development. Female flies were exposed daily or every two days to a partially intoxicating dose of ethanol for two weeks. After the exposure period, they were allowed to lay eggs for 3-5 days in the absence of any further intoxications, and their progeny was allowed to develop under standard (ethanol-free) conditions. The progeny from flies that were intoxicated daily remained active for several minutes longer than progeny from naïve parents, revealing a strikingly higher tolerance to ethanol in their first exposure to the drug. Thus, our observations indicate that prolonged exposure to ethanol can somehow imprint tolerance to the drug in the fly gametes. In addition, daily ethanol exposure of the parents caused a change in the cell composition of their intestinal epithelium, which is consistent with alterations in the turnover of their intestinal stem cells. Therefore, ongoing experiments in our lab address whether the imprinting of the gametes may have occurred during gametogenesis or at the level of the germline stem cells from which they originate.
This work was supported by a CSUN start-up grant and a CSUPERB New Investigator Award.
Poster #: 124
Campus: CSU Fresno
Poster Category: Other
Keywords: flavonol, prostate cancer, medicinal chemistry
Project Title: Studies on the Potential of 3-O-Substituted-Flavonols as Anti-Prostate Cancer Agents
Author List:
Li, Xiang; Graduate, Department of Chemistry, California State University, Fresno, Presenting Author, Eden Award Finalist
Lee, Maizie; Undergraduate, Department of Chemistry, California State University, Fresno, Presenting Author
Chen, Guanglin; Staff, Department of Chemistry, California State University, Fresno
Zhang, Xiaojie; Graduate, Department of Chemistry, California State University, Fresno
Fong, Ryan; Clovis High School
Gonzalez, Aaron; Bullard High School
Chen, Qiao-Hong; Department of Chemistry, California State University, Fresno
Abstract: Flavonoids are a class of polyphenolic compounds ubiquitously distributed in a variety of dietary plants with an array of biological activities. Flavonols are a sub-class of flavonoids featuring a hydroxyl group at C-3. Certain flavonols, such as myricetin and fisetin, have been evidenced by in vitro cell-based and in vivo animal experiments as potential anti-prostate cancer agents. However, the moderate potency and poor bioavailability have hindered their further advancement as chemotherapeutic agents. The objective of this study is to explore the possibility of enhancing anti-proliferative potency by chemical manipulations of 3-OH in flavonols. Three flavonols, 3′,4′-dimethoxyflavonol, 3′,4′,7-trimethoxyflavonol, and 3′,4′,5′-trimethoxyflavonol, were selected as our three lead compounds. Over forty derivatives of these lead compounds have been successfully synthesized through aldol condensation, Algar-Flynn-Oyamada (AFO) reaction, and alkylation. Their structures were characterized by interpreting the 1H and 13C NMR spectra. Their anti-proliferative activity toward three human prostate cancer cell lines has been assessed by WST-1 proliferation assay. The effects of one optimal derivative on PC-3 prostate cancer cell apoptosis and cell cycle progression were assessed by flow cytometric analysis. Our findings indicate i) that 3-O-alkyl-flavonols and 3-O-aminoalkyl-flavonols are more potent than the corresponding lead compound in suppressing prostate cancer cell proliferation, ii) that incorporation of a dibutylamine group to 3-OH through a three- to five-carbon linker leads to the optimal derivatives with up to 292-fold enhanced potency as compared with the corresponding lead compound, and iii) the most potent compound can activate PC-3 cell cycle arrest at the G2/M phase and promote PC-3 cell apoptosis. This project was financially supported by CSU-Fresno. A. Gonzalez was supported by ACS seed project 2016.
Poster #: 125
Campus: CSU Channel Islands
Poster Category: Other
Keywords: Nesseria Meningitidis, Nucleoside analogues, antibacterial agents
Project Title: Inhibition of Neisseria meningitidis by 2′,3′-O-isopropyldiene modified nucleosides
Author List:
Jacobsen, Casey; Undergraduate, Chemistry, California State University Channel Islands, Presenting Author
Maldonado, Jessica; Undergraduate, Chemistry, California State University Channel Islands, Presenting Author
Abstract: Nesseria meningitidis is a fatal human pathogen that causes death by inducing sepsis and of meningococcal disease. We report herein the synthesis of nucleoseide analogs incorporating the 2′,3′-O-isopropyldiene group, and the antibacterial properties of these analogs against Neisseria meningitidis and other types of bacterial infections. Three gram positive and three gram negative bacteria including, Staphylococcus aureus, Escherichia coli, Bacillus cereus, Enterococcus faecalis, Proteus vulgaris, and Neisseria meningitides were examined in this study. The growth of the N. meningitidis was selectively inhibited by the exposure to six different nucleoside analogs all containing the 2′,3′-O-isopropyldiene group. Three of the six compounds were also modified on the 5’ position by tetrachlorophthalimido group that was introduced by modified Gabrial-Mitsunobu reaction, or by an azido group. The percent inhibition includes: 5’ hydroxyl of Adenyl (10%), Urydyl (8%), and Guanylyl (9%) 2′,3′-O-isopropyldiene analogs; 5’ tetrachlorophthalimido groups on adenyl (12%) and uridyl (9%) analogs; and 5’ azido on the guanylyl (10%) analog. The percent inhibition was confirmed by disc diffusion, and brought forward to quantitative susceptibility determination. These data confirmed that 2′,3′-O-isopropyldiene group and their derivatives provide consistent selective inhibition against Nesseria meningitides.
Poster #: 126
Campus: CSU Stanislaus
Poster Category: Product-focused Innovation
Keywords: Probiotics, Lactobacillus helveticus KII13, tripeptides
Project Title: Characterization of a Novel Probiotic strain Lactobacillus helveticus KII13
Author List:
Shahbaz, Lersa Sayyad ; Undergraduate, Biological Sciences, California State University, Stanislaus, Presenting Author
Torres, Marivel; Undergraduate, Biological Sciences, California State University, Stanislaus, Presenting Author
Ah Yo, Alexis; Undergraduate, Biological Sciences, California State University, Stanislaus
Savala, Torivo ; Undergraduate, Biological Sciences, California State University, Stanislaus
Abstract: High cholesterol and hypertension are risk factors for cardiovascular disease, which remains one of the largest causes of death worldwide. Statin drugs and angiotensin-converting enzyme (ACE) inhibitors are commonly prescribed for lowering cholesterol levels and blood pressure, respectively. With society becoming more health-conscious and seeking more natural remedies for such conditions as hypertension and high cholesterol, there is an increasing interest in healthy dietary products such as probiotics. Probiotics are live microorganisms that provide health benefits when taken in appropriate doses. Lactobacilli bacteria are common probiotic strains.
Recently, we have isolated a probiotic strain of Lactobacillus helveticus KII13 that produces higher level of bioactive tripeptides (isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP)) that have anti-hypertension activity compared with other L. helveticus strains. The long-term objective of this project is to develop a freeze-dried yogurt product with L. helveticus KII13. Freeze-drying not only preserves yogurt, but also helps maintain a sufficient quantity of viable probiotics. The short-term objective is to determine how L. helveticus KII13 produces more antihypertensive tripeptides than the reference strain, L. helveticus R0052.
To understand how L. helveticus KII13 produces higher level of tripeptides, we sequenced its genome and compared it with that of L. helveticus R0052 strain. We found seven proteases/peptidases that are different between the two and thus may be responsible for the higher production of bioactive tripeptides in L. helveticus KII13. We have so far cloned genes for Peptidase CE (Pep CE) and Aminopeptidase E (Pep E) from both L. helveticus KII13 and L. helveticus R0052. We are currently expressing these genes in E. coli DE3. Once enzymes are purified, we will quantify tripeptides after incubating each enzyme with casein by using UPLC-MS/MS in Dr. Joowon Suh lab in Korea (Myongji University, South Korea).
Poster #: 127
Campus: CSU Fullerton
Poster Category: Product-focused Innovation
Keywords: cancer, ATP, signal transduction
Project Title: Identification of a Novel Group of Small Molecules as Inhibitors of β-Catenin Pathway, Mitochondrial Membrane Potential and Cell Growth
Author List:
Salhotra, Ankit; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Adkins, Lauren; Graduate, Biological Science, California State University, Fullerton
Ha, Nhi; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Hanna, Luke; Undergraduate, Chemistry and Biochemistry , California State University, Fullerton
Jelowicki, Aneta; Undergraduate, Chemistry and Biochemistry , California State University, Fullerton
Nguyen, Romie; Graduate, Chemistry and Biochemistry , California State University, Fullerton
Guzman, Stacy; Undergraduate, Chemistry and Biochemistry , California State University, Fullerton
de Lijser, Peter; Chemistry and Biochemistry , California State University, Fullerton
Patel, Nilay; Biological Science , California State University, Fullerton
Abstract: The Wnt/β-catenin canonical pathway is a well-characterized signaling pathway that plays a role in cell proliferation and cell fate decisions. In the canonical pathway, presence of Wnt prevents the formation of the destruction complex, allowing for the accumulation of cytoplasmic β-catenin. β-catenin is then free to translocate to the nucleus and interact with the TCF/LEF family to transcribe downstream genes involved in cell proliferation. Mutations in this pathway that lead to an increased level of β-catenin, are often associated with cancer. Thus, manipulation of β-catenin levels may serve as a potential anti-cancer therapeutic strategy. We designed and synthesized a library of >400 compounds to modulate the Wnt/β-catenin pathway, and screened for their effect on HeLa cell proliferation using CyQUANT assay. Some of the most potent compounds reduced cell proliferation rate by >40%. Immunocytochemistry (ICC) showed that the compounds reduced β-catenin levels in HeLa cells compared to vehicle (0.1% DMSO) treated cells. This reduction of β-catenin was more prominent in tightly clustered cells. Enzyme-linked immunosorbent assay (ELISA) confirmed that some of the active compounds reduced β-catenin levels to 30% of vehicle-treated HeLa cells. Expression of β-catenin-regulated cell cycle genes c-MYC, CCND1 and CCNB1 was attenuated to 61%, 43% and 81%, respectively, of control. Expression of CDKN1A, which is an inhibitor of cell cycle, was increased two-fold. Surprisingly, these compounds also lowered and altered Mitotracker staining, which is indicative of reduced mitochondrial membrane potential. However, inhibition of Mitotracker signal does not seem to be correlated with cell proliferation, and suggests that we may be able to design compounds that inhibit cell proliferation without affecting Mitotracker signal. Future research will determine if inhibition of Wnt/β-catenin pathway is sufficient for their anti-proliferative effects.
Poster #: 128
Campus: CSU San Marcos
Poster Category: Programmatic (Core, Stem Cell, Bridges, PSM)
Keywords: Stem cells, Liver disease,
Project Title: Using Patient Specific IPSCs to Model Liver Disease in ZZ-Mutations of Alpha-1 Antitrypsin (AAT)
Author List:
Marsolais, Renee; Undergraduate, Biology, California State University San Marcos, Presenting Author
Abstract: AAT deficiency is a potentially fatal disorder characterized by the accumulation of AAT in the liver. The most severe type (the ZZ mutation) affects an estimated 1 in 3,000, although it remains severely under-diagnosed. This disorder shows large clinical heterogeneity: despite having the same mutation, the severity of liver disease experienced by patients will vary significantly; some will require liver transplants early on, while others may experience no liver issues whatsoever. Therefore there must be some modifier, either environmental or genetic that predisposes only some patients to develop severe liver disease. We hypothesized that abnormalities of the autophagy pathway may lead to abnormal clearance and buildup of mutant AAT in affected hepatocytes. A related prediction is that abnormal autophagy correlates with degree of AAT-related liver disease, therefore supporting a role of autophagy as a clinically relevant genetic modifier in AAT deficiency. To test our hypothesis, we reprogrammed three different types of patient fibroblasts: AAT-ZZ patients with liver disease, AAT-ZZ patients without liver disease, and normal MM genotype cells as a control. We differentiated patient-specific induced pluripotent stem cells to hepatocyte-like cells (HLCs) that resemble immature hepatocytes by a standard protocol. We have evidence that AAT-ZZ patients with liver disease show a lower baseline level of autophagy when compared to AAT-ZZ patients without liver disease. Our data also raises the important possibility that ZZ HLCs from patients with severe liver disease have a blunted response to drugs that are known to induce the autophagy pathway. We are currently testing drugs that enhance the autophagic process with a focus on those that are being used or proposed as a therapy for AAT-related liver disease and measuring proteins such as LC3 that will help us define autophagy level and progression. Additionally, we are interested in learning what other environmental or genetic factors influence the presentation and progression of AAT-related liver disease in affected individuals. The results of our experiments will provide a more thorough understanding of how mutant AAT and autophagy interact and influence each other and could eventually lead to the development of new treatments for affected individuals.
Poster #: 129
Campus: CSU East Bay
Poster Category: Programmatic (Core, Stem Cell, Bridges, PSM)
Keywords: Project-Based Learning, Science Education, Molecular Biology
Project Title: CRISPR in the Classroom!
Author List:
Gallegos, Maria; Biology, Presenting Author
Abstract: Advanced Molecular Techniques (Biol 6141) is a discovery-based lab course offered at California State University, East Bay (CSUEB). This course has three aims: 1) to promote the “Vision of Change” mandate to replace cookbook-type lab courses with those that are discovery-based; 2) to teach a variety of molecula techniques applicable to academia and industry and 3) to develop molecular tools useful to the research community at-large. I first taught this course in 2007. In 2012, 56 past students of Biol 6141 co-authored a peer-reviewed publication entitled, “The C. elegans Rab Family: Identification, Classification and Toolkit Construction“. Inspired by my sabbatical at UCSF, I decided to revamp the course this year. We are now using modern molecular techniques (including site directed mutagenesis and Gibson assembly) to build molecular tools designed to edit the C. elegans genome using the CRISPR/Cas9 platform. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. Cas9 is an RNA-guided endonuclease. The CRISPR/Cas9 system functions as an acquired immune system in prokaryotes to protect against infection by foreign DNA (i.e. bacteriophages) but has been modified to work as a fast, accurate and efficient tool for genome editing.
For my first time teaching the new curriculum, I had twelve students work on plasmid construction for six unique CRISPR projects. Three projects were completed by the end of the quarter and one set was injected into C. elegans to produce a genome-edited strain. Importantly, the assigned CRISPR projects involved genes critical to my research. Thus, this strain will now be utilized by both graduates and undergraduates in my research lab to test specific hypotheses about this gene’s role in neurodevelopment. The other CRISPR projects that are in various stages of completion will also be used to initiate additional research projects in the lab.
At the CSU Annual Biotechnology Symposium, I will present the organization of the course, student work in addition to the benefits and challenges of implementing a research-based lab course. I will focus in particular on strategies that I have developed over the past 9 years that have helped me manage numerous research projects at the same time and have coerced my students to become more successful in a wet lab.
Poster #: 130
Campus: CSU San Marcos
Poster Category: Proteins (Include Proteomics)
Keywords: Amyloid, Curli, Protein-Protein Interactions
Project Title: Can CsgE Interact with Proteins not Related to Curli Formation?
Author List:
Oliver, Samantha; Undergraduate, Chemistry and Biochemistry, California State University San Marcos, Presenting Author
Tran, Amy Nhung; Undergraduate, Chemistry and Biochemistry, California State University San Marcos, Presenting Author
Binmahfooz, Ashwag; Graduate, Chemistry and Biochemistry, California State University San Marcos
Jayasinghe, Sajth; Chemistry and Biochemistry, California State University San Marcos
Abstract: Background: Curli , are a type of cell surface filament found on enteric bacteria such as E. Coli and Salmonella. These filaments are thought to play an important role in host cell adhesion and invasion. Assembly of curli involve six proteins: CsgA, CsgB, CsgC, CsgE, CsgF, and CsgG. Out of these, CsgF and CsgE are thought to play an important role in the construction of the curli fibers. CsgE has been shown to prevent the in vitro aggregation of CsgA and is thought to function as a chaperone protein in vivo. The nature of the CsgE-CsgA interaction has not been characterized, and it is not known if CsgE can interact with other proteins. In this study we sought to determine the ability of CsgE to interact with lysozyme, a protein unrelated to cluri formation, but with a molecular weight similar to that of CsgA, in order to investigate the specificity of CsgE-protein interactions. Methodology: A BL21 (DE3) bacterial expression system was used to express four different single cysteine mutants of CsgE (34C, 58C, 76C, and 99C where the wild type residue at the respective position was replaced by a cysteine). After expression of the protein in the presence of IPTG purified protein was obtained using Ni affinity chromatography, and the purity determined using SDS-PAGE. The single cysteine residue on each mutant was derivatized with the environment sensitive fluorescent probe IAEDANS and purified again using Ni affinity chromatography. Interaction between CsgE and lysozyme was investigated using fluorescence quenching in the presence of the fluorescence quencher acrylamide to determine the accessibility of the IAEDANS fluorophore to quencher with and without lysozyme. Results and Discussion: In the absence of lysozyme we obtained Stern-Volmer quenching constants of 0.770, 0.695, and 0.938 for CsgE labeled with IAEDANS at positions 34, 58, and 99 respectively. IEADANS labeled at these three positions exhibited quenching constants of 0.284, 0.299, and 0.591 in the presence of lysozyme. This reduction in the quenching constant suggests that the accessibility of acrylamide to the fluorophore is reduced in the presence of lysozyme. Based on this observation we postulate that CsgE is not limited to binding proteins that are involved in curli formation.
Poster #: 131
Campus: CSU Fresno
Poster Category: Proteins (Include Proteomics)
Keywords: cAMP receptor protein, DNA binding, Gly184
Project Title: Gly184 is critical for the function of cAMP receptor protein from Escherichia coli
Author List:
Chand, Shristi; Graduate, Biology, California State University, Fresno, Presenting Author
Kanwar, Parinav; Undergraduate, Biology, California State University, Fresno, Presenting Author
Sangha, Navjot; Undergraduate, Biology, California State University, Fresno
Hicks, Matt; USDA at Parlier
Youn, Hwan; Biology, California State University, Fresno
Abstract: The Escherichia coli cAMP receptor protein (CRP) utilizes the helix-turn-helix motif for DNA binding. The latter recognition helix of CRP, termed F-helix, is composed of six amino acid residues: Arg180, Glu181, Thr182, Val183, Gly184 and Arg185. These residues are in direct contact with target DNA. Among them, three residues (Arg180, Glu181 and Arg185) are generally known to be important for CRP’s DNA-binding function. However, little is known about the role of the other three residues. Here we show that Gly184 is another critical residue for CRP function. Experimentally, we randomized the codon for Gly184. The randomized plasmid pool was then introduced into an E. coli CRP reporter strain, and the resultant transformants were screened for active phenotypes. Subsequent analyses revealed that only Gly (wild type residue at position 184) could provide an in vivo transcriptional activity to the level of wild type CRP. There was also a second tier of a few amino acids at position 184 which yielded measurable transcriptional activity, albeit not the level of wild type CRP; these amino acids included Ser, Thr, Tyr, Ala and Cys. Wild type CRP and these CRP mutants were purified and their in vitro DNA-binding affinity was measured using a fluorescence polarization method. Surprisingly, G184S and G184Y mutants, which were impaired in terms of transcriptional activity, showed a wild-type level DNA-binding activity. Our results suggest that Gly184 may have dual roles in CRP function, one for DNA binding and the other for proper interaction with RNA polymerase. The current working hypothesis for the importance of Gly184 is that only Gly184 can confer flexibility to the F-helix which is required for both proper DNA binding and interaction with RNA polymerase.
Poster #: 132
Campus: CSU Northridge
Poster Category: Proteins (Include Proteomics)
Keywords: RGS proteins, NMR spectroscopy, protein dynamics
Project Title: Using NMR to probe molecular motions of regulator of G protein signaling 7 (RGS7) in its interactions with Ga(i1)
Author List:
Jimenez, Julie; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Crowhurst, Karin; Chemistry and Biochemistry, California State University, Northridge
Abstract: G protein signal regulation is essential in neurotransmitter signaling. Misregulation of this system has been linked to several neurological diseases such as depression, epilepsy and Parkinson’s. The signaling process is modulated by regulators of G protein signaling (RGS proteins). RGS proteins are responsible for speeding up the deactivation of G proteins associated with G protein-coupled receptors (GPCRs) by expediting the hydrolysis of the alpha subunits of G proteins, thus arresting the signal. Each RGS protein contains a 130 “box” domain that binds the alpha subunits of G proteins. Within the RGS family we study RGS4 and RGS7; their binding sites vary by only 2 residues. RGS4 targets Ga(i1), while RGS7 targets primarily Ga(o) but also binds Ga(i1) with lower affinity. The selection process for this binding event is not entirely understood but we hypothesize that protein motions (not just the amino acid sequence) contribute to binding and selectivity.
Proteins are dynamic biomolecules whose motions (dynamics) are well-suited for study using nuclear magnetic resonance (NMR) spectroscopy. We postulate that protein motions contribute greatly to the selective interaction between RGS7 and Ga(i1). 1H 15N correlation spectra of unbound and Ga(i1)-bound RGS7 were recorded. When overlaid and compared the spectra revealed widespread shift of peaks as well as the appearance of new peaks in the bound spectrum. One normally expects chemical shift changes corresponding to residues solely in the binding interface of a protein. The extensive chemical shift changes and the manifestation of new peaks hint at allosteric processes taking place. We are therefore interested in measuring and comparing backbone and sidechain protein motions of unbound and Ga(i1)-bound RGS7 using NMR. Currently we are working on chemical shift assignments of side chain methyl groups in preparation for analyzing dynamics data. This work has provided evidence that protein dynamics contribute to the binding of RGS7 to Ga(i1). Further investigation of these protein motions that contribute to binding is crucial for improved understanding of the selectivity of a particular RGS for specific G proteins. This may ultimately aid in the design of useful medication to combat various neurological diseases.
We sincerely thank the NSF for funding our research and the purchase of our NMR spectrometer (MCB-1158177 and CHE-1040134).
Poster #: 133
Campus: CSU Bakersfield
Poster Category: Proteins (Include Proteomics)
Keywords: Cancer, Lysyl Oxidase, Protein Chemistry
Project Title: Inhibition of Cancer Cell Viability Using Lysyl Oxidase Inhibitors
Author List:
Johnston, Kathryn; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield, Presenting Author
Lopez, Karlo; Chemistry and Biochemistry, California State University, Bakersfield
Abstract: Lysyl oxidase (LOX) is a copper-dependent amine oxidase that has been implicated in playing a paradoxical role in cancer. The enzyme’s main role is to catalyze the crosslinkages in collagen and elastin, and has also been implicated in the proliferation of tumors in late-stage cancer. As a result, lysyl oxidase in breast cancer cells has been selected as a target for inhibition. It was hypothesized that the activity of LOX in cancer cells can be controlled through the use of the mechanism-based inhibitor 2-aminopropionitrile (β-APN). MDA-MB-231 breast cancer cells were used to test derivatized forms of this inhibitor. In particular, the meta and para derivatives of 4-nitrobenzyl β-APN, the para derivative of 4-bromobenzyl β-APN, the dibenzyl derivative of β-APN, and the 3,3-((4-bromobenzyl)azanediyl)dipropanenitrile were tested by means of viability assays. These assays revealed that cells treated with the meta derivative of 4-nitrobenzyl β-APN and the para derivative of 4-bromobenzyl β-APN had a significant decrease in cell viability when inhibitor concentrations greater than 500 μM were used. The para derivative of 4-nitrobenzyl β-APN, however, had little effect on the viability on the cells. These results indicate that the meta derivative of 4-nitrobenzyl β-APN and the para derivative of 4-bromobenzyl β-APN are successfully targeting the cancer cells.
In order to test whether the inhibitors were selectively targeting lysyl oxidase, western blot analyses using anti-LOX antibodies were carried out and fluorometric activity assays using Amplex red were performed. These assays showed that lysyl oxidase was present in cancer cells in large quantities and that the inhibitors had an effect on the activity. As a control, normal cells were also tested by western blot analysis and fluorometric activity assays were carried out. The controls showed that lysyl oxidase was present in normal cells as well, but to a lesser degree and was almost 3.5 times less active than in cancer cells. Given these data, it is proposed that lysyl oxidase is a good candidate for inhibition with these small-molecule β-APN derivatives.
Poster #: 134
Campus: Sacramento State University
Poster Category: Proteins (Include Proteomics)
Keywords: apolipoprotein , amyloid, protein folding
Project Title: Reversibility and nucleation potential of acid-induced aggregation in apolipoprotein A-I
Author List:
Nguyen, Chau; Undergraduate, Chemistry, California State University, Sacramento, Presenting Author
Tran, Thao; Graduate, Chemistry, California State University, Sacramento
Roberts, Linda; Chemistry, California State University, Sacramento
Abstract: Cardiovascular disease is the number one cause of death throughout the world. Apolipoprotein A-I (apoA-I) is the major protein in high density lipoprotein (HDL) whose function is to remove excess cholesterol from peripheral tissues and transport it to the liver for excretion, thus lowering the risk for heart disease. Plasma apoA-I on its own is correlated with lowered risk for heart disease. Despite its cardioprotective properties, apoA-I, either by mutation or certain environmental conditions, forms amyloid deposits in a variety of tissues including cardiac and vascular tissue. Acidic pH, which can occur in microenvironments during many physiological processes, destabilizes proteins causing partial unfolding that could lead to amyloid structure. We have investigated the effect of a wide range of pH’s on the formation of aggregates in apoA-I and have found that pH 4-5.5 stimulates rapid aggregation of the protein. The isoelectric point of apoA-I is ~4, thus at least some of the aggregation could be due to simple isoelectric point precipitation. Such precipitation should be reversible and unable to seed aggregation of normally folded protein at neutral pH. In this research project, we examined 1) the reversibility of the aggregation at pH 4 upon restoration to neutral pH and 2) the ability of acid-induced aggregates to seed aggregation of protein at neutral pH.
Acid-induced aggregation of WT apoA-I was produced by combination of stock protein in phosphate buffered saline (PBS) at pH 7 with McIlvaine’s buffer to a final protein concentration of 0.1 mg/mL and a pH of 4.0. After 30 minutes, insoluble aggregates were isolated by centrifugation for 15 minutes. Aggregation was detected by measuring light scattering at 340 nm. Light scattering intensity of apoA-I at pH 4 prior to centrifugation was 0.8. The intensity of insoluble aggregates resuspended at pH 7 was 0.02. Addition of 0.1 mg/mL protein at pH 7 to insoluble aggregates slightly raised the intensity to 0.07. The high reversibility of acid-induced aggregation and the lack of ability to seed aggregation at neutral pH together suggest that the aggregates formed at pH 4 may be due at least partly to isoelectric point precipitation rather than amyloid formation. To differentiate between amyloid and amorphous aggregation, future work will examine the binding of the amyloid specific dye thioflavin T to apoA-I at pH 4.
Poster #: 135
Campus: CSU Fullerton
Poster Category: Proteins (Include Proteomics)
Keywords: tetrahydrofolate, tetrahydromethanopterin, diacylglycerol kinase
Project Title: Evolutionary relationships among dihydrofolate and dihydromethanopterin reductase homologs in methylotrophic α-proteobacteria
Author List:
Burton, Mark; Graduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Abanobi, Chidinma; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Wang, Tzu-Chi; University of California, Berkeley (Post-baccalaureate Research Assistant)
Rasche, Madeline; Chemistry and Biochemistry, California State University, Fullerton
Abstract: Microbial one-carbon (C1) metabolism involves the use of either tetrahydrofolate (H4F) or tetrahydromethanopterin (H4MPT). The latter coenzyme was thought to be exclusive to the Archaea domain until the discovery of H4MPT-linked C1 transfer enzymes in the α-proteobacterium Methylobacterium extorquens AM1. This organism contains two protein sequences annotated as dihydrofolate reductases (DfrA and DfrB) and one called dihydromethanopterin reductase (DmrA). The three proteins share 50%, 25%, and 26% sequence identity, respectively, with bacterial Dfrs, but only the function of DmrA has been demonstrated biochemically. Moreover, sequence alignment searches using BLAST have recognized scores of proteins that share up to 99% sequence identity with DmrA, and yet are currently annotated as diacylglycerol (DAG) kinases. In this work, we have used enzymatic assays and bioinformatics analysis to provide insight into the biochemical function and phylogeny of selected DmrA homologs, currently annotated as either Dfrs or DAG kinases. Despite sharing 34% sequence identity with DmrA, DfrB was unable to reduce a dihydromethanopterin analog (dihydrosarcinapterin, H2SPT); however, DfrB could effectively reduce dihydrofolate with a specific activity of 3.2 U · mg protein-1, comparable to the rate measured for Escherichia coli Dfr (8.2 U · mg-1). In contrast, the Hyphomicrobium nitrativorans DmrA homolog (69% identity) annotated as a DAG kinase was shown to reduce archaeal H2SPT with a Km of 30 μM H2SPT and a Vmax of 12 U · mg-1. These values are close to those obtained for M. extorquens DmrA (Km of 66 μM H2SPT, Vmax of 6.6 U · mg-1). Furthermore, the annotated DAG kinase was incapable of phosphorylating 1,2-dihexanoyl-sn-glycerol when compared to commercial DAG kinase from E. coli. Phylogenetic tree analysis showed DfrA and DfrB clustered outside the clades of DmrA homologs labeled as DAG kinases. These results provide the first biochemical evidence that DfrB functions as an authentic dihydrofolate reductase, and indicate that DAG kinases with greater than 65% identity to DmrA may be mis-annotated and instead are likely to function in tetrahydromethanopterin biosynthesis.
This research was funded by National Science Foundation grant CHE-1508801 and a CSUPERB Faculty-Student Collaborative Research: Development Grant.
Poster #: 136
Campus: CSU Fresno
Poster Category: Proteins (Include Proteomics)
Keywords: YdeH, Escherichia coli, diguanlyate cyclase
Project Title: Purification and characterization of YdeH, a diguanylate cyclase, and its zinc-site mutant
Author List:
Zhou, Yue; Graduate, Biology, California State University, Fresno, Presenting Author
Montiel, Christian; Undergraduate, Biology, California State University, Fresno
Saunders, Cameron ; USDA at Parlier
Toride, Moeko; Graduate, Chemistry, California State University, Fresno
Brooks, Cory; Chemistry, California State University, Fresno
Youn, Hwan; Biology, California State University, Fresno
Abstract: Cyclic di-GMP is a second messenger which controls important cellular processes in bacteria such as biofilm formation and virulence. YdeH is one of the 14 Escherichia coli proteins containing a GGDEF motif, which is known to be essential for the diguanylate cyclase activity of synthesizing cyclic di-GMP from GTP. The crystal structure of YdeH shows that the enzyme is composed of an N-terminal zinc-binding domain and a C-terminal catalytic domain. There is in vivo evidence that the activity of YdeH is negatively regulated by zinc binding. Here, we purified both wild type YdeH and a zinc-site mutant, C52A YdeH, and monitored the effect of zinc on their in vitro enzyme activities. A histag was attached to the C-terminus of wild type and C52A YdeH, and therefore nickel affinity chromatography was used for protein purification. The purity of each protein was > 90% homogeneity based on SDS-PAGE. Both proteins were then assayed using a standard reaction mixture, but in the absence or presence of zinc (1 µM, 10 µM, 100 µM and 1 mM). After the reactions, the amounts of cyclic di-GMP produced were measured using a C18 column attached to an Agilent 1100 HPLC system. The specific activity was defined as pmol cyclic di-GMP produced per µg protein. In the absence of zinc, wild type YdeH and the C52A mutant displayed a similar specific activity, which may suggest that wild type YdeH was isolated as zinc-free apo form. In the presence of zinc, both wild type and C52A YdeH lost their enzymatic activity in a concentration-dependent manner, confirming the negative role of zinc. However, there was no significant difference in the amount of activity loss between wild type and C52A YdeH (the activity of wild type YdeH was inhibited at a slightly lower zinc concentration and therefore at a slightly faster rate). This is unexpected, because C52A YdeH has a substitution at one of four zinc binding residues (His22, Cys52, His79, His83), so it would have significantly decreased zinc affinity. This may imply either (i) inhibition by zinc is indirect, not by binding to the structurally-identified site or (ii) C52A substitution may be corrected by an unknown, adventurous residue. To test the latter possibility, we are currently constructing double mutants of YdeH (C52A/H22A, C52A/H79A and C52A/H83A) which will then be purified and characterized.
Poster #: 137
Campus: CSU San Marcos
Poster Category: Synthetic Chemistry
Keywords: Organocatalysis, Bisphosphorylimides, Friedel–Crafts
Project Title: Chiral Bisphosphorylimides as Organocatalysts for Friedel–Crafts Reactions
Author List:
Ahlberg, Lina; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author
Thompson, Cristofer; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author
Dean, Melisa; Undergraduate, Chemistry & BIochemistry, California State University San Marcos
Diaz, Gloria; Undergraduate, Chemistry & BIochemistry, California State University San Marcos
Ghasb, Elie; Undergraduate, Chemistry & BIochemistry, California State University San Marcos
Klasic, Brittney; Undergraduate, Chemistry & BIochemistry, California State University San Marcos
Maynard, Jessica; Undergraduate, Chemistry & BIochemistry, California State University San Marcos
Robinos, Jacqueline; Undergraduate, Chemistry & Biochemistry, California State University San Marcos
Abrous, Leila; Postdoc, Chemistry & Biochemistry, California State University San Marcos
Iafe, Robert; Chemistry & BIochemistry, California State University San Marcos
Abstract: As an ever-increasing number of chiral bioactive molecules flood the pharmaceutical industry, the importance of efficient enantioselective synthesis and purification of these compounds also increases. Enantioselective organocatalysis has emerged as a powerful synthetic tool complementary to traditional metal-containing catalytic transformations. The use of organocatalysts are attractive because reactions tend to be operationally simple due to their stability to air and water, ready availability, and low toxicity. Taking this into account, we have developed the asymmetric Brønsted-acid catalysis of the Friedel–Crafts reaction of sesamols, a naturally occurring component of sesame oil that shares the chemical structure of many pharmaceutical compounds, with benzylidenecarbamates using chiral bisphosphorylimides as an organocatalyst. Despite their notable potential for catalysis, bisphosphorylimides are currently underutilized in this role. The chemical scaffold of bisphosphorylimides is highly structured and ordered, mimicking the active site of an enzyme. The long-term objective is the enantioselective synthesis of bioactive natural products, unnatural amino acids, and other various building blocks useful in the synthesis of chiral bioactive molecules.
Results: An improved synthetic route of the model bisphosphorylimide organocatalyst (38% yield over 2 steps) from enantiopure BINOL using liquid ammonia has been developed in our laboratory. Catalyst activity of the model bisphosphorylimide was optimized using sesamol with a benzylidenecarbamate electrophile (Ar = tolyl) and triethyl amine as an additive at reduced temperature to afford the target amine in quantitative yield in 1.5 hours. Catalyst loading is effective as low as 5 mol %, and nucleophilic weak bases hinder the rate of reaction. The bisphosphorylimide also shows catalytic ability with two other benzylidenecarbamate electrophiles: Ar = phenyl (45% yield) and Ar = anisyl (65% yield). Furthermore, two derivatives of the organocatalyst with bulky aromatic substituents (R = phenyl, triphenylsilyl) have been prepared using the Suzuki reaction in high yields (96% yield) and fully characterized. These bulky catalysts are currently being used to determine the substrate scope of the reaction.
Acknowledgements: We thank CSUPERB (New Investigator) for support of this work
Poster #: 138
Campus: CSU Bakersfield
Poster Category: Synthetic Chemistry
Keywords: Organic, beta-aminoproprionitrile, Lysyl oxidase
Project Title: Synthesizing Small Lysyl Oxidase Inhibitors with β-aminoproprionitrile
Author List:
Williams, Meagan; Undergraduate, Chemistry & Biochemistry, California State University, Bakersfield, Presenting Author
Kamel, Joy; Undergraduate, Chemistry & Biochemistry, California State University, Bakersfield, Presenting Author
Solano, Danielle; Chemistry & Biochemistry, California State University, Bakersfield
Abstract: Lysyl oxidase (LOX) is a protein partly responsible for cancer metastasis when induced by hypoxic tumor cells. Studies show that treating these cells with β-aminopropionitrile (β-APN) actually inhibits the LOX enzyme which, in turn, prevents the metastasis of the cancerous tumor. Unfortunately, β-APN is toxic and kills epidermal cells, therefore ingestion of it is harmful. This study focuses on synthesizing molecules that will attach to the β-APN, in order to focus its toxicity only to cancer cells, all while still allowing the β-APN to inhibit LOX and only be released once in contact with the hypoxic tumor. Target molecules are synthesized by the reductive amination of aldehydes and β-APN, which are a series of 3-(benzylamino)propionitrile products. So far, the aldehydes that have been reacted with βAPN are 4-bromobenzaldehyde, 4-nitrobenzaldehyde, and 2-methoxy-4-nitrobenzaldehyde. The product of β-APN and 4bromobenzaldehyde showed the best inhibition, contrary to the belief that nitro benzaldehydes would make the best inhibitors. Given these results, the aldehydes that will be reacted with βAPN are benzaldehyde, 2-bromobenzaldehyde, 3-bromobenzaldehyde, and 4-methylbenzaldehyde. The newly synthesized products will then be tested against the LOX enzyme to determine which inhibitors are most effective, in order to continuously improve upon the results of the study, in an attempt to make the most effective inhibitors. Of the molecules that were tested (4-bromobenzaldehyde, 4-nitrobenzaldehyde, and 2-methoxy-4-nitrobenzaldehyde), it was hypothesized that the poor reactive quality of the bromine functional group makes 4-bromobenzaldehyde the best carrier molecule for the βAPN. This instilled the idea that the synthesis of benzaldehydes with variously positioned bromine substituents, along with other less reactive substituents such as methyl groups is the best course of action for producing the most effective LOX inhibitors. Acknowledgements: Funding: NSF MRI 1229301, Louis Stokes Alliance for Minority Participation (LSAMP), Crowdfunding REU, CSUPERB, the SRS grant, and the TSSR. Collaboration: Dr. Lopez’ Biochemistry Lab, especially Kathryn Johnston.
Poster #: 139
Campus: CSU Northridge
Poster Category: Synthetic Chemistry
Keywords: terpyridine, synthesis, cancer
Project Title: Preparation of terpyridine based anti-tumor agents
Author List:
Weeks, Cal; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Rptchian, Nazaret; Graduate, Chemistry and Biochemistry, California State University, Northridge
Kelson, Eric; Chemistry and Biochemistry, California State University, Northridge
Abstract: 2,2′:6′,2″-Terpyridines (Terpys) and their ruthenium complexes have been reported as promising anti-tumor agents that interfere with DNA replication and transcription (likely through intercalation, the insertion into the base-pair stack) and exhibit selectivity toward tumor cells. Despite this promise, Terpys have not been widely explored due to synthetic difficulties. Recently, we have developed methodologies to prepare polypyridines with a wide range of modifications. The goal of this work was to apply and expand these techniques to prepare Terpys with polyaromatic appendages chosen to improve DNA intercalation and incorporate these into ruthenium complexes. 1-Naphthyl and 9-phenanthryl boronic acids reacted with 4’-chloro-Terpy and potassium and cesium carbonates in DMF with standard Suzuki coupling catalysts to form NapTerpy or PhanTerpy, respectively. This was extended to the preparation of TerpyTerpy through the tandem borylation of 4’-chloro-Terpy and subsequent Suzuki coupling to another equivalent of 4’-chloro-Terpy in DMF with standard catalysts. Unfortunately, the tandem borylation of 4-chloro-Terpy and Suzuki coupling to bromophenanthrolines failed, and bromophenanthrolines themselves would not borylate under normal conditions. Efforts are underway to employ borylation/coupling promotors and other solvent systems to these reactions. The ruthenium complexes (NapTerpy)Ru(DMSO)Cl2 and (PhanTerpy)Ru(DMSO)Cl2 (where DMSO is dimethylsulfoxide) were prepared from the reaction of the Terpys with Ru(DMSO)4Cl2 in refluxing DMF. [(Terpy)Ru(TerpyTerpy)]Cl2 was prepared from the slow addition of (Terpy)Ru(DMSO)Cl2 to a suspension of TerpyTerpy in refluxing DMF. While the ruthenium complexes were reasonably soluble in aqueous solutions, substituted Terpys were often sparingly soluble. Aqueous solutions of transition metal ions including Zn(II), Fe(II), Ni(II), Cu(II), Ag(I), and Au(I) were found to dramatically improve the solubility of substituted Terpys likely through the loose (and reversible) formation of complexes. Overall, this work has produced a range of useful customized Terpy anti-tumor candidates for testing for DNA binding and tumor culture toxicity. We thank CSUPERB (Research Development Grant) and the NIH (SCORE S06 GM48680) for support of this research.
Poster #: 140
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Kinase Inhibition, Organocatalysis, Atropisomerism
Project Title: Atroposelective Synthesis of Common Structural Motifs in Medicinal Chemistry
Author List:
Maddox, Sean; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Ayonon, Arianna; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Abstract: Kinases are critical in cell signaling, protein regulation, etc. Although each kinase plays a specific role in these pathways, the active sites of the kinases are highly conserved throughout the known kinome, making it difficult to selectively inhibit or tag a specific kinase. With regards to drug discovery, this lack of selectivity often leads to several off target effects, which have the potential to cause negative side effects when treating disease.
Atropisomerism is a form of chirality, which differs from normal point chirality in that the chirality is about a bond (i.e. aryl-aryl bonds), hence racemization can spontaneously occur via bond rotation. The vast majority of small molecule kinase inhibitors contain one or more axes of chirality, giving rise to atropisomerism. Our lab has recently exploited this stereochemical phenomenon to increase kinase inhibitor selectivity. We took a promiscuous parent pyrrolopyrimidine (PPY) kinase inhibitor and rigidified its atropisomeric axis, rendering each enantiomer atropisomerically stable at physiological conditions. The resulting stable enantiomers were not only more selective, they were also found to possess activity towards different kinases, effectively decoupling the activity of the rapidly racemizing parent compound. However, the synthesis of these selective inhibitors is hindered by the reliance on semi-preparative chiral HPLC, severely limiting the scalability and efficiency of our synthesis.
To avoid this bottleneck, we have since focused our efforts toward the synthesis of atropisomerically pure PPY kinase inhibitors. One strategy to overcome this problem is through the nucleophilic Dynamic Kinetic Resolution (DKR) of rapidly racemizing atropisomers. We have found cinchona alkaloid catalysts can efficiently catalyze the nucleophilic addition of thiophenol into the quinone moiety of a rapidly interconverting PPY – quinone scaffold, affording atropisomerically stable PPY – 1,4-diol scaffolds in high yields and enantiomeric ratios (i.e. 98% yield, 98:2 enantiomeric ratio). This is an unprecedented approach towards the enantioselective synthesis of atropisomers with diverse substitution patterns. Furthermore, we have extended this DKR to the enantioselective synthesis of other common structural motifs such as diaryl ethers, a first-in-class transformation. This new catalytic methodology holds the potential to increase the efficiency of synthesizing enantiomerically pure kinase inhibitor analogs.
Poster #: 141
Campus: CSU Chico
Poster Category: Synthetic Chemistry
Keywords: organic synthesis, bifunctional organic linker molecules, solar PV cell
Project Title: Synthesis of Linker Molecules Connect Nanomaterials for solar PV Cells
Author List:
Melchor, Daniela; Undergraduate, Chemistry and Biochemistry, California State University, Chico, Presenting Author
Juette, Eugene; Undergraduate, Chemistry and Biochemistry, California State University, Chico, Presenting Author
Sajben, Nick ; Undergraduate, Chemistry and Biochemistry, California State University, Chico
Fang, Yichen; Pleasant Valley High School
Herrera, Xochith; Undergraduate, Chemistry and Biochemistry, California State University, Chico
Pu, Ying-Chih; University of California Santa Cruz
Ma, Haixia; University of California Santa Cruz
Zhang, Jin Z.; University of California Santa Cruz
Li, Yat; University of California Santa Cruz
Zhang, Jinsong; Chemistry and Biochemistry, California State University, Chico, Presenting Author
Abstract: Every year the demand for electricity increases exponentially. As a result, the field of solar energy grows, and the necessity to find ways of increasing efficiency expands. Previous research experiments have proved that metal oxide (MO) nanocrystalline materials paired with quantum dots (QD) showed great potential that could satisfy the demand of cost-efficient energy. A fundamental understanding of the interplay between materials properties and their photovoltaic (PV) performance is critical and transformative for developing new nanocomposite PV materials. Attempts at connecting QD’s and MO’s with bifunctional organic linker molecules were successful, linker molecules with the general form X-R-Y (X =-SH, -NC, -CH=CH2 and other soft ligands and Y = -COOH, -OH, -NH2 and other hard ligands) were synthesized. The QD-linker-MO assembly was constructed and the charge transfer efficiency was observed to have been amplified by 3 to 4 times more compared to directly depositing QD’s to MO’s with absence of the linker molecules. Schematic synthesis of the linker molecules with a general formula of HS-R-COOH, required an acylation, Newman-Kwart Rearrangement followed by hydrolysis.
Via the synthesis of organic aromatic linker molecules, further experimentation showed that the Newman-Kwart Rearrangement activation energy is affected by the substituent on the aromatic structure. The strength of the electron withdrawing substituent decreased the temperature and time required. It was observed in p-nitrophenol that the Newman-Kwart Rearrangement reaction, could be bypassed during the acylation stage of the schematic. The development of converting C-O bond to C-S bond in one-pot instead of a traditional 3-step reaction will be reported. The method developed in the research group has an average yield of 90% compared to an overall 20% yield for the three step reaction. This development provides the synthetic community an important tool to make C-S bond, which will be widely used in synthesizing compounds with biological activities.
The poster will report the following:
1. Synthesis of linker molecules;
2. Assembly of the linker molecules with the nanomaterials;
3. Study of electron transfer efficiency including SEM images, optical properties, PEC performance;
4. Novel C-O to C-S conversion that hasn’t been presented in other conferences
This work was supported by a CSUPERB Research Development grant. A manuscript is under preparation.
Poster #: 142
Campus: Sacramento State University
Poster Category: Synthetic Chemistry
Keywords: glycodendrimer, organic synthesis, green chemistry
Project Title: A Green Divergent Synthesis of a Hexavalent Glycodendrimer Using Cellobiose
Author List:
Dimas, Dustin ; Graduate, Department of Chemistry, California State University, Sacramento, Presenting Author
McReynolds, Katherine; Department of Chemistry, California State University, Sacramento
Abstract: All dendrimers have common characteristics: A core, linkers that increase the number of ends, and terminal functional groups where the chemistry can occur. For glycodendrimers, the functional groups can include amino, carboxyl or aminooxy moieties. In our research, the dendrimers terminate in aminooxy groups, which can then react chemoselectively with sugars to yield oxime-linked glycodendrimers. As dendrimers have globular structures with multiple ends, they can exhibit the multivalent effect. Multivalency refers to the simultaneous interactions of multiple binding sites on one entity to multiple receptor sites on another. With this unique ability, sulfated glycodendrimers have been shown to bind to HIV virions and block fusion, and therefore the infection, of host cells. Our research is focused on the synthesis of multivalent glycodendrimers as HIV entry inhibitors.
In the present study, a seven-step pathway was used to synthesize a hexavalent core, which was then used to create a cellobiose-terminated glycodendrimer. The first step in the process was to make the linker. The first reaction involved the addition of an aminooxy group on one end of the diol, diethylene glycol (DEG). A Mitsunobu reaction was first accomplished, with a yield of 67%. Next, a methanesulfonyl group was added to the remaining OH to complete the linker synthesis with a yield of 55%. The second step in the process was to make a trivalent core. In the first step, triethanolamine and acrylonitrile were reacted to create a nitrile terminated core with a yield of 88.1%. The next step was a two-step, one pot reaction to reduce the nitrile group and protect the primary amine group with a tert-butyloxycarbonyl (BOC) group, with a yield of 64.7%. Next, the BOC group was removed with trifluoroacetic acid (TFA) and dichloromethane with a yield of 86.1 %. To make the hexavalent core, the mesylated linker was added to the tri-amine core under SN2 conditions with a yield of 34%. To complete the synthesis of the hexavalent core, hydrazinolysis will be performed, yielding the desired aminooxy-terminated hexavalent core. In the final step, cellobiose will be added to the hexavalent core. This reaction will be conducted in a 400 W microwave (CEM MARS 5) to yield the desired oxime-linked cellobiose-terminated glycodendrimer. Further research will be performed to optimize the above steps, and finally to assess to the anti-HIV activity of the sulfated products.
Funding: NIH:1SC3GM119521-01.
Poster #: 143
Campus: San José State University
Poster Category: Synthetic Chemistry
Keywords: trifluoromethylation, light-driven hydroxylation, catalysis
Project Title: Tandem C—H Functionalizations Via Light-Driven Biocatalysis and Photocatalysis
Author List:
Sosa, Victor; Graduate, Chemistry, San José State University, Presenting Author
Melkie, Marya; Undergraduate, Chemistry, San José State University, Presenting Author
Faris, Justin; Undergraduate, Chemistry, San José State University
Kato, Mallory; Staff, Chemistry, San José State University
Cheruzel, Lionel; Chemistry, San José State University
Abstract: The development of convenient synthetic methods for green and sustainable synthesis of pharmaceutical and other chemicals has become an increasing research topic. Selective functionalization of unactivated C—H bonds is an important, yet difficult synthetic step, to achieve that would afford new synthetic strategies for the production of valuable synthons. We are reporting herein on a tandem C—H functionalization approach utilizing visible light activation and the Ru(II)-diimine complexes. Such complexes have unique photochemical properties and are involved in single electron transfer steps.
Our laboratory initially developed hybrid P450 enzymes containing a Ru(II)-diimine photosensitizer covalently attached to the P450 heme domain in order to perform P450 reactions upon visible light excitation. The photosensitizer is used to provide the necessary electrons to the heme domain in order to activate molecular dioxygen and sustain photocatalysis in the selective hydroxylation of several natural substrates with high total turnover numbers. A combination of protein engineering approaches has then enabled the selective light-driven hydroxylation of a variety of non-natural substrates including mono-substituted arenes. Recently, we sought to implement photoredox catalysis in our lab with the light-driven trifluoromethylation (CF3) of arenes using the same Ru(II) complexes. The trifluoromethyl functionality is a crucial group in agrochemicals, material science, and in medicinal chemistry. In pharmaceuticals, the presence of a CF3 group has been shown to enhance binding selectivity, increase of lipophilicity and improves metabolic stability. We utilized photoredox trifluoromethylation to functionalize various mono and disubstituted arenes. After column and high performance liquid chromatography (HPLC) purification, the trifluoromethylated products were thoroughly characterized by 1H, 13C and 19F NMR spectroscopy and gas chromatography/mass spectrometry (GC/MS). These newly generated CF3 products were then further selectively hydroxylated by the light-driven hybrid enzymes.
Overall, combining the photoredox trifluoromethylation with the light-driven P450 enzymatic hydroxylation has yielded, in two light-driven steps, valuable sought-after synthons towards the synthesis of pharmaceuticals.
Poster #: 144
Campus: CSU San Marcos
Poster Category: Synthetic Chemistry
Keywords: Gold(I) catalysis, Friedel–Crafts, microwave
Project Title: Gold(I)-Catalyzed Friedel–Crafts-like Arylation of Benzylic Alcohols to Afford 1,1-Diarylmethanes
Author List:
Oakley, James; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author
Stanley, Tyler; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author
Iafe, Robert; Chemistry & Biochemistry, California State University San Marcos
Abstract: As a relative newcomer to the field of homogeneous catalysis in organic chemistry, gold catalysts have demonstrated high catalytic activity in numerous organic transformations. The structural motifs attainable by these methods are a common building block for the synthesis of several natural and synthetic products ubiquitous in biologically active compounds. For instance, several potent anticancer, antidepressant, antifungal, anti-inflammatory, antimicrobial, antitumor, antiviral (including antiretroviral), and cytotoxic agents can be synthesized from gold catalysis. During our prior investigation using gold(I) salts to catalyze etherification reactions, we discovered that phenols react via a Friedel–Crafts reaction mechanism instead of the etherification pathway. To our satisfaction, we were successfully able to cleanly promote the Friedel–Crafts reactivity using microwave conditions. From this, our lab has developed the first microwave-assisted, gold(I)-catalyzed Friedel–Crafts reaction of electron-rich benzenes with benzylic alcohols to afford 1,1-diarylmethanes, a common structural motif found in many pharmaceuticals including breast cancer treatment drugs. To our knowledge, while examples of gold(III)-catalyzed Friedel–Crafts reactions are well-known, a microwave assisted, gold(I)-catalyzed Friedel–Crafts reaction is currently unprecedented in the literature. The long-term objective is the synthesis of bioactive natural products and other various building blocks useful in the synthesis of bioactive molecules
Results: The gold(I)-catalyzed Friedel–Crafts reaction has been optimized using sesamol as the nucleophile, 5 mol % of the gold(I) and silver(I) salts, and slightly elevated temperatures to afford 98% of the target 1,1-diarylmethane. Using these conditions, the substrate scope for this reaction currently includes the reaction of the following electron-rich nucleophiles in excellent yields: 1-naphthol, 2-naphthol, p-methylanisole, and p-tert-butylphenol. Various electron-rich arenes are currently being explored as well as an intramolecular capstone example to synthesize fluorenol derivatives in high yields.
Poster #: 145
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: fluorescence, organic, DNA
Project Title: Monitoring DNA duplex formation with chemically modified fluorescent cytidine analogues
Author List:
Burns, Dillon; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Teppang, Kristine; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Lee, Raymond; Graduate, Chemistry and Biochemistry, San Diego State University
Lokensgard, Melissa; Graduate, Chemistry and Biochemistry, San Diego State University
Purse, Byron; Chemistry and Biochemistry, San Diego State University
Abstract: Fluorescent nucleosides are an invaluable tool for monitoring biochemical processes involving nucleic acids, including DNA and RNA. Studying the how the microenvironment of DNA changes during binding events can be challenging, but fluorescent nucleosides are tools that illuminate these changes. Prior to our studies, the toolbox of fluorescent nucleosides has not included molecules that are able to report on the formation of double-stranded DNA by increasing the brightness of their fluorescence. Previously studied fluorescent nucleosides, such as tricyclic cytidines tC and tCo maintain their bright fluorescence in DNA, but there is no turn-on response. In this work, we aimed to test the hypothesis that a new, electronically modified tC could give us this turn-on response. We used a sequence of synthetic reactions to prepare a new fluorescent probe, 8-DEA-tC, that is modified to impart a push-pull character to its electronic structure. This molecule was synthesized using aryl substitution reactions to create a fluorescent nucleobase, which was then ribosylated using a chemically activated 2′-deoxyribose compound to create the new nucleoside, a synthetic building block for DNA. We then used solid-phase DNA synthesis to prepare short DNA segments containing the molecule and fluorescent spectroscopy to measure the photophysical properties. Our results show that 8-DEA-tC is able to respond to DNA duplex formation with up to a 20-fold increase in fluorescence, by a wide margin the most powerful such effect known. Mechanistic studies of this fluorescence increase will inform the rational design of the next generation of fluorescent nucleobase analogues.
Poster #: 146
Campus: CSU San Bernardino
Poster Category: Agriculture/Biofuels/Environment
Keywords: bioinformatics, Solanum lycopersicum, RNA-Seq
Project Title: Identification of Long Non-Coding RNAs and Alternative Splicing Events During Tomato Fruit Development
Author List:
Morikone, Michael; Undergraduate, Computer Science & Engineering, California State University, San Bernardino, Presenting Author
Zheng, Yi; Postdoc, Boyce Thompson Institute
Fei, Zhangjun; Boyce Thompson Institute
Abstract: Long non-coding RNAs (lncRNAs) are a class of regulatory RNA that are longer than 200 base pairs and do not code for protein. Alternative splicing (AS) is a process in gene transcription where a multi-exon gene is spliced into two or more different mature transcripts. Solanum lycopersicum, a model plant for fleshy fruit development, was studied through the use of RNA-Seq analysis for lncRNAs and AS events in order to better understand the molecular mechanisms of fleshy fruit development. Five distinct stages of tomato fruit with three biological replicates were used to monitor the changes in cell expanison, maturation, and ripening. The purpose of this project was to generate a comprehensive list of isoforms from the RNA-Seq data in order to identify lncRNAs, AS events, as well as new protein-coding genes. It was determined from this dataset that there were 565 lncRNAs with 162 of these having significant changes in expression during fruit development. It was also shown that 289 paired isoforms had significant differential expression between stages. Additionally, there were 214 new protein coding genes that were identified from the dataset. Novel results include a newly discovered lncRNA that was identified which is an example of a cis-natural antisense transcript and showed an inverted expression pattern to a zeta-carotene isomerase gene, a key component of lycopene biosynthesis, suggesting a possible regulatory role of this lncRNA in carotenogenesis. Future work will include investigation of these novel results from both bioinformatics and molecular biology approaches.
Poster #: 147
Campus: CSU East Bay
Poster Category: Agriculture/Biofuels/Environment
Keywords: iTRAQ, phosphoproteomics, white lupin
Project Title: Quantitative phosphoproteomics to unravel white lupin’s signal transduction in response to phosphorus deficiency
Author List:
Amadi , Michael; Graduate, Biological Sciences, California State University, East Bay, Presenting Author
Cole, Jason; Undergraduate, Biological Sciences, California State University, East Bay
Li, Kathy; UCSF
Chalkley, Robert; UCSF
Burlingame, Al; UCSF
Uhde-Stone, Claudia; Biological Sciences, California State University, East Bay
Abstract: Phosphorus (P) availability is one of the most limiting factors for crop production worldwide. To breed or engineer crops that do well in poor soils, researchers are investigating plants that are well adapted to P deficiency, such as the legume white lupin (Lupinus albus L.). While great progress has been made in understanding white lupin’s strategies to acquire P, not much is known about the signal transduction pathways that link sensing of P deficiency with the appropriate responses. Phosphorylation of proteins plays a central role in signaling processes. Proteins that change phosphorylation status when exposed to P deficiency are potential members of such signal transduction pathways. However, quantifying the dynamics of protein phosphorylation is challenging, because of their transient nature and low abundance. A sensitive method is needed to identify proteins that become phosphorylated after exposure to P deficiency.
To this end, white lupin was grown in hydroponics with sufficient phosphorus (+P). After 3 weeks, half of the lupin plants were transferred into P-deficient nutrient solution (-P). After 24 hours, total protein was isolated from roots of two +P and two –P24h plants, for a total of two independent biological replications. Proteins were quantified, trypsin-digested, and subjected to LC-MS (Liquid chromatography-mass spectrometry). This successfully identified hundreds of white lupin proteins, indicating good quality of sample preparation. We then used four different iTRAQ (isobaric tags for relative and absolute quantification) tags to differentially label the +P and –P peptides from the two replicates. We are currently in the process of enriching phosphorylated peptides by titanium dioxide chromatography. Next, we plan to perform multiplex UPLC-MS/MS (Ultra-Performance Liquid chromatography tandem mass spectrometry) to quantify the iTRAQ-labeled phosphorylated peptides from +P and –P24h in 3 biological replicates. Peptides that are not phosphorylated under +P, but become phosphorylated after transfer to –P are likely members of white lupin’s signal transduction pathway in response to P deficiency.
Poster #: 148
Campus: CSU Monterey Bay
Poster Category: Agriculture/Biofuels/Environment
Keywords: bioreactor, bioremediation, agricultural runoff
Project Title: An Examination of Nitrate Concentrations on Bacterial Denitrification Rates in Woodchip Bioreactors
Author List:
Mortensen, Zane; Undergraduate, School of Natural Sciences, California State University, Monterey Bay, Presenting Author
Leandro, Mason; Undergraduate, School of Natural Sciences, California State University, Monterey Bay, Presenting Author
Carpenter, Alyson; Undergraduate, School of Natural Sciences, California State University, Monterey Bay
Ball, Alexandra; Undergraduate, School of Natural Sciences, California State University, Monterey Bay
Haffa, Arlene; School of Natural Sciences, California State University, Monterey Bay
Silveus, John; School of Natural Sciences, California State University, Monterey Bay
Abstract: California is the leading agricultural state with 13% of the US market. This level of productivity requires the addition of fertilizers resulting in nutrient-rich agricultural effluent. Bioremediation using woodchip bioreactors is a promising management practice for mitigation of excess nutrient runoff. To explore the efficacy of woodchip bioreactors, faculty and student researchers at CSU Monterey Bay have created a field laboratory utilizing bacterial communities genetically selected for their ability to denitrify agricultural effluent. Optimum bacterial denitrification requires a diverse and healthy bacterial community, as well as specific environmental conditions to facilitate the metabolic processes of mesophilic bacteria including pH, temperature, dissolved oxygen, and an adequate source of both nitrogen and carbon. While monitoring bacterial population and denitrification rates in a controlled environment, nitrate levels were adjusted to address specific research questions about denitrification rates and bacterial concentrations. Experiments with the bioreactors under increased nitrate addition indicate a decrease in the rate of denitrification and a decline in bacterial concentrations. Published research suggests bacterial denitrification rates in woodchip bioreactors decreases in relation to increased nitrate concentrations as carbon availability becomes the limiting factor. Continuing research will investigate carbon supplementation to improve the bacterial colonization and therefore the effectiveness of denitrification in woodchip bioreactors under increased nitrate concentrations.
Poster #: 149
Campus: CSU Fresno
Poster Category: Agriculture/Biofuels/Environment
Keywords: herbicide resistance, fleabane, genetics
Project Title: Investigating the genetic basis of glyphosate resistance in the San Joaquin Valley agricultural weed hairy fleabane (Erigeron bonariensis)
Author List:
Molina, Rigoberto; Undergraduate, Biology, California State University, Fresno, Presenting Author
Pathak, Ankit; Graduate, Biology, California State University, Fresno
Waselkov, Katherine; Biology, California State University, Fresno
Abstract: Agricultural weeds are a major problem for U.S. farmers, with efforts to control these pests costing an estimated $20 billion per year. For perennial crops in which tillage is infrequent, herbicides are the most effective tool for weed control, and the most widely applied herbicide across the U.S. is glyphosate. This broad-spectrum, low-toxicity herbicide has been so widely applied in the Central Valley of California since the 1990s that several weed species of orchards and vineyards have evolved resistance to it. Among these weeds is Erigeron bonariensis, hairy fleabane, with glyphosate resistance (GR) detected in California in 2007. As yet, the genetic mechanism of this resistance is unknown, but preliminary studies have indicated that target-site mutations in the coding sequence of the enolpyruvyl-shikimate synthase (EPSPS) enzyme affected by glyphosate, and non-target-site mutations in other genes or regulatory elements, can both be involved in glyphosate resistance. Research in the related species horseweed (E. canadensis) has yielded several candidate genes for glyphosate resistance in the genus Erigeron. Our objective for this project is to investigate possible genetic changes underlying target and non-target resistance mechanisms in a single well-characterized GR population of hairy fleabane. Thus far, we have completed our investigation of the target-site mechanism. Plants were greenhouse-grown from seed collected from a field-verified GR fleabane population and a glyphosate sensitive (GS) population, and tissue was collected from eight plants of each biotype. DNA was extracted and PCR was performed to amplify selected exons from three candidate genes for Sanger sequencing: EPSPS-1, and ABC transporter class C variant M7 and variant M10. Results showed that EPSPS-1 did not have any of the commonly-observed target site mutations for GR, but several nonsynonymous mutations were observed in the M7 and M10 ABC transporter genes, which are implicated in non-target-site resistance. We conclude that non-target resistance mechanisms such as impaired glyphosate translocation and/or upregulation of the EPSPS gene may underlie the GR in fleabane. The next step in this project will be to employ quantitative real-time PCR of the three candidate genes with glyphosate-treated GR fleabane plants to test this hypothesis. Our research will provide insight into the evolution of glyphosate resistance and help design new strategies for weed control.
Poster #: 150
Campus: Sacramento State University
Poster Category: Agriculture/Biofuels/Environment
Keywords: arbuscular mycorrhizal fungi, restoration, diversity
Project Title: Diversity of arbuscular mycorrhizal fungi before and after restoration from unused agricultural soil to riparian forest
Author List:
Simonoff-Smith, Alisa; Graduate, Biological Sciences, California State University, Sacramento, Presenting Author
Todd, Austin; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Boren, Drew; Undergraduate, Biological Sciences, California State University, Sacramento
Ewing, Nicholas; Biological Sciences, California State University, Sacramento
Abstract: Due to its varied topography and climate California is one of the world’s diversity hotspots. There are 3500 species of vascular plants and 61% of these are found nowhere else in the world. California has been very active in restoration of natural habitats, especially in the Central Valley where agriculture has replaced most of the natural environment.
Mycorrhizal fungi penetrate plant roots and form symbiotic relationships. In doing this they aid in nutrient uptake, pathogen protection, soil stabilization, and seedling establishment. In exchange, they receive carbon compounds needed for growth and survival. The health of an ecosystem is dependent on the mycorrhizal partners and so there is increasing interest in understanding the role of AM fungi in restoration.
The purpose of this study is to determine the diversity of AM in an unused agricultural field near the American River Parkway in Sacramento County before and six months after restoration to a riparian forest. To accomplish this, soil was collected from 32 sites before and after restoration with half of the sites having been amended with AM during restoration. DNA isolated from soil samples was subjected to PCR to amplify an 800 bp fragment of the ribosomal small subunit that was cloned and subjected to DNA sequencing. To date we have analyzed over 650 DNA sequences from 32 soil samples (at least 20 per sample) that comprise the complete pre-restoration set of samples. We are currently completing DNA isolation, PCR, and sequence analysis for the 32 post-restoration samples. For all pre-restoration sequences, similarities to published sequences were determined using the BLAST (GenBank). Phylogenetic analysis was then carried out on our sequences and the closest matches using MEGA6. Diversity was determined by Richness and the Shannon-Weiner Index. We found ten genera of AM fungi, significantly higher than typically found in agricultural land and approaching that of natural habitats. We hypothesize that both amended and unamended sites will show lower diversity post-restoration with amended sites highest in the species used for amendment while the unamended site will be high in colonizing species. These results have implications for successful restoration efforts of agricultural land.
This project has been funded by the C.M. Goethe and Delisle Family Bequests.
Poster #: 151
Campus: CSU Northridge
Poster Category: Agriculture/Biofuels/Environment
Keywords: microbiome, nematodes, bacteria
Project Title: ¬16S rDNA Sequence Analysis of the Bacterial Communities in Decomposing Beetles from Los Angeles
Author List:
Samayoa, Briana; Undergraduate, Biology, California State University, Northridge, Presenting Author
Ocfemia, Maria; Undergraduate, Biology, California State University, Northridge
Isip, Jonah; Undergraduate, Biology, California State University, Northridge
Patel, Dhrumi; Undergraduate, Biology, California State University, Northridge, Presenting Author
Flores, Gilberto; Biology, California State University, Northridge
Hong, Ray; Biology, California State University, Northridge
Abstract: Advances in high-throughput DNA sequencing of ecological samples have enabled unprecedented insight into host-microbe associations. However, the microbes mediating the interaction between insects and nematodes– possibly the most species-rich and ubiquitous animal phyla on Earth– have received paltry attention. To address this deficit, we surveyed bacterial composition of the scarab beetles in greater Los Angeles in the summer of 2015. Necromenic nematodes infect specific beetle species and wait for the hosts to die before resuming development by feeding on the microorganisms on the decaying cadaver. Our previous findings indicate that each beetle species has a unique bacterial microbiome (the aggregate of bacteria that exist on the surface and inside a host), but this beetle microbiome was not reflected by the nematode’s bacterial microbiome. However, beetle-derived bacteria appear to be more nutritious: we isolated a novel strain of Citrobacter that can accelerate nematode development compared to the conventional but artificial E. coli OP50 diet used in laboratories. Using 16S rDNA high-throughput sequencing on the Illumina MiSEQ platform, we tested the hypothesis that Cyclocephala and Serica beetles differ in their microbiome profiles when they were alive but perhaps due to bias in the decomposing environment, the bacterial community decreased in species diversity under aerobic conditions from day 2 to 10. We found that although there is a loss of species diversity in decomposing beetles, there are also distinct community profiles according to the age of the carcass. This approach will enable us to discover which subset of bacteria from the beetle microbiome is actually consumed by the nematodes and thus potentially form the basis for their interactions.
Poster #: 152
Campus: CSU Long Beach
Poster Category: Agriculture/Biofuels/Environment
Keywords: Glycoside Hydrolase, Metagenomics, Biofuel
Project Title: Glycoside Hydrolase Multidomain architecture is affected by the environment
Author List:
Nguyen, Stanley; Graduate, Biological Sciences, California State University, Long Beach, Presenting Author
Berlemont, Renaud; Biological Sciences, California State University, Long Beach
Abstract: Glycoside hydrolases (GHs) produced by microbes are involved in the breakdown of complex polysaccharide (e.g., cellulose, chitin) in the environment and are important enzymes for biotechnology (e.g., biofuel). Across ecosystems, the distribution of GH enzymes matches the carbohydrate supply. Interestingly GHs are sometime associated with accessory non-catalytic domains such as carbohydrate binding modules (CBMs) aimed at anchoring the catalytic domains (i.e., GHs) to their substrate. These multidomain enzymes display reduced diffusion and improved catalytic efficiency, however it has been shown in vitro that when water availability decreases, the catalytic benefit associated with CBMs decreases. Here we tested the effect of the ecosystem type on the frequency of CBMs relative to the frequency of GHs. Specifically, we hypothesized that CBM/GH would be low in environments with reduced water content (e.g., soil, skin) and high in liquid environments (e.g., marine and gut ecosystems). We tested this hypothesis using a custom bioinformatic pipeline aimed at identifying GH and CBM domains in ~1,000 publicly accessible metagenomes corresponding to 13 broadly defined ecosystems. In liquid environments, the substrate specific CBM/GH was higher than in dry environments. This suggested that the carbohydrate supply has a strong effect on GH distribution whereas the water content strongly affects the multidomain architecture of enzymes involved in carbohydrate processing.
Poster #: 153
Campus: CSU Northridge
Poster Category: Astrobiology
Keywords: permafrost, Fluorescence Activated Cell Sorting, 16S rRNA
Project Title: Identifying Changes in the Active, Dead, and Dormant Microbial Community Structures across a Chronosequence of Ancient Alaskan Permafrost
Author List:
Burkert, Alex; Graduate, Biology, California State University, Northridge, Presenting Author
Mackelprang, Rachel; Biology, California State University, Northridge
Abstract: Microbial communities within permafrost (perennially frozen ground) survive and reproduce for millennia despite extreme conditions such as water stress, subzero temperatures, high salinity, and low nutrient availability. However, we do not fully understand how these organisms are able to survive. Understanding how life adapts to extreme conditions in terrestrial permafrost could provide clues to survival strategies for life on other frozen planetary bodies within our solar system and beyond. Previous studies have used metagenomic and 16S rRNA gene sequencing to characterize community structure and functional potential in attempts to understand how these organisms adapt to the challenges associated with long-term survival in a permanently frozen environment. However, freezing temperatures may preserve DNA from dead organisms for extended periods of time. Because metagenomic and 16S rRNA gene sequencing do not distinguish between live, dead, and dormant cells, it is difficult to determine which organisms are viable. This study focuses on developing strategies to differentiate the live, dead, and dormant populations of low biomass permafrost microbial communities. Preliminary experiments demonstrated that live and dead cells could be separated via Live/Dead staining coupled with fluorescence activated cell sorting. Further, we were able to enrich for endospores using treatments that lyse vegetative cells and we were able to inhibit PCR amplification of DNA from dead cells. These findings suggest that the combined protocols can be used to isolate the live, dead, and dormant microbial communities from low biomass environmental samples. Ongoing work focuses on applying these methods to a chronosequence (12 kyr, 25 kyr, and 35 kyr) of Pleistocene permafrost located near Fairbanks, Alaska. The goal of this project is to sequence the 16S rRNA gene of the live, dead, and dormant cell populations to observe changes over geologic time. Funding has been provided by NASA, CSUN Associated Students, CSUN Graduate Studies, and CSUPERB. Additional acknowledgements include Rachel Mackelprang, Gilberto Flores, Kerry Cooper, Mark Waldrop, Tom Douglas, as well as the members of the Mackelprang Lab.
Poster #: 154
Campus: CSU Fullerton
Poster Category: Astrobiology
Keywords: Flow chemistry, Prebiotics, Organic synthesis
Project Title: Continuous flow synthesis of 2-amino-oxazole and subsequently ribo/arabino furanosyl amino-oxazolines from prebiotic precursors.
Author List:
Kading, Joshua; Undergraduate, Chemistry and Biochemistry., California State University, Fullerton, Presenting Author
Abstract: Glycolaldehyde dimer and cyanamide have been shown to be the first compounds that were necessary for the formation of DNA/RNA nucleosides cytidine, uridine, adenosine and guanosine. When glycolaldehyde and cyanamide are exposed to certain conditions, they react to form 2-amino-oxazole. 2-amino-oxazole is the chemical precursor to nucleoside base pairs:[1] when 2-amino-oxazole is exposed to (DL)-glyceraldehyde it can cyclize to form a precursor to (DL)-ribose/deoxyribose, the backbone of DNA and RNA. Batch/flask conditions do not offer optimized production of 2-amino oxazole and its derivatives. We report here that the continuous flow synthesis of 2-amino-oxazole under ambient temperatures and pressures using water produces a mean yield of 67.8% ± 8.9%. Higher temperatures and pressures do not appear to impact yields; however, the presence of a base such as NaOH or KH2PO4 can significantly alter yields; when the precursors were mixed in the presence of 0.25 M NaOH the yield was optimized to 80% and higher, promoting elimination that is essential in the final mechanistic step. Subsequently, reacting 2-amino-oxazole with (DL)-glyceraldehyde in water forms the ribo/arabino furanosyl amino-oxazolines in yields ranging from 29 to 68 %, depending on experimental flow conditions.
Poster #: 155
Campus: CSU San Bernardino
Poster Category: Astrobiology
Keywords: Thermophile, Cultivation, Archaea
Project Title: Cultivation of a member of Group 4 Aigarchaeota from thermophilic cellulolytic consortia
Author List:
Gonzalez, Cristina; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Miranda, Michelle; Undergraduate, Biology, California State University, San Bernardino
Gonzalez, Lizett; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Sam, Christian; Undergraduate, Biology, California State University, San Bernardino
Dodsworth, Jeremy; Biology, California State University, San Bernardino
Abstract: Aigarchaeota is a thermophilic lineage within in the domain Archaea that does not have any cultivated representatives. Based on phylogenetic inferences, Aigarchaeota might represent a phylum or a deeply branching member of the Thaumarchaeota within TACK super phylum, which has been proposed to be a sister lineage to the progenitor of eukaryotes. Group 4 Aigarchaeota (Aig G4), one of nine genus level groups that have been detected so far, was previously found to be enriched in lignocellulose (corn stover) in situ enrichments at an ~85 °C site in Great Boiling Spring (GBS), NV. To attempt to cultivate Aig G4 in the laboratory, twelve anaerobic enrichment media conditions were designed and inoculated using Aig G4 corn stover in situ enrichments in the field. Maintenance of Aig G4 in enrichment cultures was assessed by quantitative polymerase chain reaction using primers specific for a region of the Aig G4 16S rRNA gene. Aig G4 was successfully maintained at 1-5% of total 16S rRNA gene copies and 0.5–2×106 16S rRNA gene copies/mL in two enrichment cultures. These cultures were grown in anaerobic conditions at 80 °C on semi-synthetic media containing GBS spring water with corn stover, keratin, and xyloglucan as major growth substrates and either HEPES or bicarbonate as buffers. Further experiments determined that corn stover, but neither keratin nor xyloglucan, could serve as a sole substrate to support growth of Aig G4. No growth of Aig G4 was observed under microaerophilic conditions, suggesting that it may be strictly anaerobic. No growth was observed in the presence of 0.1 mM thiosulfate and nitrate, suggesting that one or both of these compounds are toxic to Aig G4. Aig G4 were not maintained in synthetic medium without spring water or supplemented with a bulk organic extract derived from GBS spring water, suggesting that one or more inorganic components of GBS water may be required for growth. Fluorescence in situ hybridization (FISH) was also used to visualize the presence and morphology of Aig G4 in the in situ corn stover enrichments. These laboratory enrichment cultures will serve as a basis for testing substrate utilization of Aig G4 using stable isotope labeling experiments and FISH-nanoSIMS. Furthermore, studying the growth of Aig G4 and other thermophilic microbes on corn stover will help us better understand the degradation of cellulosic material at high temperatures.
This work was supported by NSF grant DEB-1557042.
Poster #: 156
Campus: CSU Long Beach
Poster Category: Astrobiology
Keywords: astrobiology, hypersaline, virus
Project Title: Physico-chemical investigation of GNphi2, an extremely hypersaline Halorubrum virus
Author List:
Laporte, Richer; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Dillon, Jesse; Biological Sciences, California State University, Long Beach
Sabet, Shereen; Staff, Biological Sciences, California State University, Long Beach
Abstract: Extremely halophilic Bacteria and Archaea are capable of surviving environments of high salt concentrations up to 35% (350 grams salt per liter). These halophiles are found in the Great Salt Lake, the Dead Sea, solar salterns, as well as in ancient salt deposits. These hypersaline habitats are similar to those that have recently been identified on Mars, making halophiles a good model system for the search for present or past life on the “Red Planet.” Many halophiles have been successfully utilized in medicinal, agricultural, and environmental purposes including vaccine development, increasing salt toleration of crops, and degradation of hydrocarbons. Hypersaline viruses have also been isolated from extremely hypersaline environments, although far fewer of these have been characterized compared with their prokaryotic hosts. This study is focusing on the characterization of a hypersaline virus, called GNphi2, isolated from the Exportadora de Sal (ESSA) salterns in Mexico that infects a Halorubrum (haloarchaeal) host. GNphi2 belongs to the Myoviridae family of viruses and has a dsDNA genome that is approximately 82 kb in size, which codes for a DNA polymerase, helicase, and a pseudogene replicase. To understand its potential in biotechnological applications, we first wanted to characterize the range of physico-chemical conditions under which the halovirus remains infectious, so, we subjected the virus to different salinities (0% to 35%) and temperatures (-80oC to 80oC), and then measured their lytic activity via top agar plaque assays. We have discovered that GNphi2 is still infectious between 30-60oC, but not at or above 70°C. For the salinity experiments, we saw that no plaques were formed at 0% salt, but GNphi2 is still highly lytic over a broad range of salinities, between 5% and 35%. Ongoing experiments are addressing lower temperatures, as well as tolerance to different levels of pH and desiccation. This study is advancing our understanding of extremophilic viruses that can possibly inform the search for life on Mars. Furthermore, since these viruses are able to function in extreme salinity, then potential biotechnological applications such as their use as shuttle vectors to transfer haloadaptive genes from slower growing halophiles to faster growing, mesophilic bacteria, such as E. coli, to induce salt tolerance and improve efficiency in various industrial or commercial processes.
Poster #: 157
Campus: CSU Northridge
Poster Category: Astrobiology
Keywords: permafrost, astrobiology, microbiology
Project Title: Carbon Metabolism in Pleistocene Permafrost Microbial Communities
Author List:
Mahendrarajah, Tara; Graduate, Biology, California State University, Northridge, Presenting Author
Mackelprang, Rachel; Biology, California State University, Northridge
Abstract: Permafrost is gaining interest as a model for astrobiology. Since six of the other eight planets in our solar system, as well as their moons, asteroids, and comets are permanently frozen, life on these celestial bodies is most likely to be found in a subzero environment. On Earth, life can exist in permafrost for millennia and may act as an analogue reflecting potential inhabitants on extraterrestrial cryogenic bodies. Active microbial life exits in even the most ancient permafrost, but we know little about the metabolic strategies utilized by permafrost microbes that enable survival over geologic time. Even in nutrient rich permafrost, most resources are sequestered away from the microorganisms by freezing conditions. To investigate, we sampled from a chronosequence of Pleistocene permafrost ranging in age from 12,000 – 35,000 years before present. We measured extracellular enzyme activity specific to carbon metabolic processes using a fluorometric enzyme assay. Across all age categories, microbial communities were capable of metabolizing various carbohydrate and amino acid substrates. Highly active enzymes included those involved in the degradation of cellulose. In general, enzymatic activity decreased per unit mass in older samples. These data likely reflect lower cell counts in older samples and may also point to decreased cellular activity. Current studies are underway to determine how biomass differs across the chronosequence and how enzyme activities relate to the amount of biomass in permafrost.
Poster #: 158
Campus: Cal Poly Pomona
Poster Category: Astrobiology
Keywords: astrobiology, planetary protection, biodegradation
Project Title: Biodegradation of Kleenol-30 by Acinetobacter radioresistens 50v1, a Mars Odyssey-associated microorganism
Author List:
Lalla, Sidharth; Graduate, Chemistry & Biochemistry, California State Polytechnic University, Pomona, Presenting Author
Madrid, Steve; Undergraduate, Chemistry & Biochemistry, California State Polytechnic University, Pomona, Presenting Author
Barding, Gregory; Chemistry & Biochemistry, California State Polytechnic University, Pomona
Mogul, Rakesh; Chemistry & Biochemistry, California State Polytechnic University, Pomona
Abstract: The importance of proper cleaning procedures in regards to spacecraft assembly has always been of the utmost importance to NASA. To help ensure cleanliness, spacecraft are assembled in cleanrooms facilities, where benchtop surfaces are routinely wiped down with alcohols and floors cleansed with detergents such as Kleenol-30. Despite these stringent cleaning protocols, spacecraft assembly facilities contain a persistent bioburden, with the Acinetobacter being among the most abundant genera. In this poster presentation, we will provide molecular evidence the supports the biodegradation of Kleenol-30 by A. radioresistens 50v1, which is a bacterium isolated from the surface of the pre-flight Mars Odyssey orbiter. Under minimal conditions (0.2x M9, 26 µM Fe(II)), with ethanol as a sole carbon source, this bacterium degrades Kleenol-30 to yield products such as triethylene glycol and octaethylene glycol, along with other compounds (which could not be statistically identified). These biodegradation products were measured using gas chromatography-mass spectrometry, where changes in the organic content of the culture broth were measured at late-log phase. In the presence of Kleenol-30, the abundances of several bacterial metabolites from the culture broth also changed, with 1-monopalmitin, glycerol monostearate, 4-hydroxybenzoate, and 2-ketoisocaproic acid all increasing in abundance. In contrast, deconoate, oxoproline, and 2-ketoglutarate all decreased in abundance in the presence of Kleenol-30. Additionally, cultivation studies show that 0.1% v/v Kleenol-30 decreases the lag time by ~2-fold, while no impact was observed on the growth rate in the presence of 0.1% or 1.0% Kleenol-30. In conclusion, these combined results indicate that (A) A. radioresistens 50v1 endures metabolomic changes to manage the presence of Kleenol-30 and (B) suggest that growth during lag phase is enhanced by low amounts of Kleenol-30. The current status of this project will be discussed, as will the implications for planetary protection and Mars exploration.
Poster #: 159
Campus: CSU San Marcos
Poster Category: Biochemistry
Keywords: protein folding, bioconjugation, single-molecule
Project Title: Single Molecule Protein Folding at CSU San Marcos
Author List:
Howe, Jesse; Undergraduate, Chemistry and Biochemistry, California State University San Marcos, Presenting Author, Nagel Award Finalist
Walters, Gregory; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Hamadani, Kambiz; Chemistry and Biochemistry, California State University San Marcos
Abstract: Protein folding is an extremely complex, heterogeneous, dynamic, and inherently asynchronous process. Single molecule fluorescence methods provide a unique and ultrasensitive set of probes for monitoring the conformations and dynamics of protein chains within physiologically relevant contexts. Single molecule fluorescence resonance energy transfer (smFRET) in particular is capable of monitoring the distances between two fluorescent dyes with sub-nanometer spatial resolution and up to nanosecond temporal resolutions. Even so, smFRET has remained inaccessible to most biophysical researchers and especially to undergraduate research institutions due to the difficulties involved in making properly dye-labelled samples and the expense and/or complexity of existing single-molecule fluorescence detection platforms. To address this issue, we demonstrate proof-of-principle for both streamlined fully-in-vitro sample generation and smFRET detection of libraries of dual-labelled proteins and ribosome-bound nascent chains (RNCs) by undergraduate students using an inexpensive and robust home-built confocal microscope. The microscope is designed to detect freely diffusing molecules but also has a 3-axis piezo-scanning stage which enables imaging and trajectory analysis of surface immobilized molecules. Here we present our PCR-templated and highly-efficient method for expressing and dual-labelling RNCs and proteins in-vitro using a purified and reconstituted translation system together with unnatural amino acid incorporation and copper-click chemistry. This process lends itself to automated high-throughput sample generation as well as single molecule analysis. Using such an approach large libraries of proteins can potentially be screened for single-molecule phenotypes of interest. Our methods will be used to explore how the ribosome affects protein-folding reactions.
Poster #: 160
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: apolipoprotein, HDL, antimicrobial
Project Title: Binding interaction of lipid-bound apoA–I with lipopolysaccharides and phosphatidylglycerol
Author List:
Haeri, Bahareh; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Weers, Paul; Chemistry and Biochemistry, California State University, Long Beach
Abstract: Apolipoprotein A-I (apoA-I) is a major component of high-density lipoprotein (HDL), an anti-antherogenic complex responsible for reverse cholesterol transport from peripheral tissues to liver, and mediates several essential metabolic functions related to heart disease. Human apoA-I is 28 kDa protein found in lipid-free and lipid-bound forms. Previous studies have shown that lipid-free apoA-I binds and neutralizes lipopolysaccharides (LPS), which are the major constituents of the outer bacterial membrane of gram-negative bacteria, therefore reducing endotoxic effects. In addition, the protein destabilizes bilayers of negatively charged phosphatidylglycerol (PG), a phospholipid found in the inner bacterial membrane which may result in cell lysis. However, the majority of apoA-I is found in the lipid-bound form as HDL; therefore binding interactions with LPS and PG were studied. To this end reconstituted HDL (rHDL) was produced with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and apoA-I, which was isolated by KBr density ultracentrifugation. rHDL was analyzed by native PAGE and showed three distinct lipid-protein complexes of 246, 203, and 149 kDa. To compare the binding of lipid-free and lipid bound apoA-I to LPS, non-denaturing PAGE was employed. This showed that LPS binding was similar for lipid-free apoA-I and rHDL. Both forms of apoA-I were able to disaggregate LPS micelles, as shown by LPS carbohydrate staining. To investigate the binding to PG, unilamellar PG vesicles with encapsulated calcein were prepared by extrusion. Calcein is a fluorescent dye and used as an indicator of lipid vesicle leakage, which has self-quenching ability at a concentration above 70 mM. Binding to the vesicles was measured by the increase in calcein fluorescence, which was released upon protein-vesicle binding. Lipid-free apoA-I induced the release of 60 to 70% calcein from the PG vesicles, however, calcein release was reduced to 20 to 30% for rHDL. These results show that when apoA-I is in the lipid-bound state, binding interaction with PG bilayer vesicles was decreased significantly, however, no effect on LPS binding was observed. Thus in order to be most effective as an antimicrobial protein, apoA-I needs to be in the lipid-free conformation. This research was supported by a grant from the National Institutes of Health (NIGMS #GM089564).
Poster #: 161
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Cross-linking, Lipoproteins, lecithin cholesterol acyltransferase
Project Title: The conformation of apolipoprotein E4 on discoidal and spherical high density lipoproteins using chemical crosslinking and fluorescence spectroscopy
Author List:
Bala, Noor; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Taiwo, Mary Kehinde; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Narayanaswami, Vasanthy; Chemistry and Biochemistry, California State University, Long Beach
Abstract:
Human apolipoprotein E (apoE) is a 34 kDa exchangeable apolipoprotein that plays a critical role in plasma and brain cholesterol transport. APOE gene polymorphism results in three common alleles ɛ2, ɛ3, and ɛ4, which produce apoE2, apoE3, and apoE4 protein isoforms, respectively. Whereas apoE3 is considered anti-atherogenic, apoE4 is an established risk factor for developing Alzheimer’s disease (AD). In the brain, apoE is mainly associated with high density lipoprotein (HDL)-like particles. An early event in HDL biogenesis is association of apoE with phospholipids and cholesterol to form discoidal particles that are remodeled into spherical HDL by lecithin cholesterol acyltransferase (LCAT). The overall objective of the study is to understand the conformational change accompanying LCAT-mediated conversion of discoidal to spherical HDL. To achieve this, seven variants of recombinant apoE4, L14C, A29C, A62C, A102C, K146C, A216C and A256C, were expressed in E. coli, purified by affinity chromatography, and reconstituted with POPC or POPC/cholesterol. Reconstituted HDL (rHDL) was isolated by density gradient ultracentrifugation and subjected to Cys-specific cross-linking using bismaleimidohexane, which bears a 13Å spacer arm. SDS-PAGE revealed the presence of 72kDa bands corresponding to dimeric apoE4 with all variants. This indicates that the Cys residues on two neighboring apoE4 molecules are ~10 Å apart on rHDL, and that two apoE4 molecules are oriented in a parallel manner around the periphery of discoidal rHDL. Recombinant human LCAT was expressed in CHO cells and its activity confirmed by fluorimetric assay and with rHDL as substrate. We are currently examining the conformation of apoE4 in spherical HDL. In addition, we will employ spatially sensitive fluorescence probes to determine proximity of Cys in the particles. It is anticipated that these independent but complementary approach will yield distance constraints that would inform us about the organization of apoE4 on discoidal and spherical HDL. The significance of this study is that it offers an innovative approach to obtain insight into the structure and organization of apoE on large lipoprotein complexes. Further, it allows us to identify potential apoE isoform-specific differences from a structural perspective and determine distinguishing features that contribute to the role of apoE4 in developing AD.
This project is supported by a grant from the National Institutes of Health #GM105561.
Poster #: 162
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Cancer, immunotherapy, ELISA
Project Title: High affinity antibody binding modulated by a size dependent conformational epitope
Author List:
Gokanapudi, Naveen; Graduate, Chemistry, California State University, Fresno, Presenting Author
Brooks, Cory; Chemistry, California State University, Fresno
Abstract: Human epithelial cells with a secretory function have a transmembrane protein called Mucin 1 (MUC 1). This glycoprotein has a highly glycosylated protein backbone consisting of a variable number of tandem repeats (VNTR). In healthy cells, the protein backbone of MUC1 is completely covered with glycosylation while the protein backbone is exposed in tumor cells due to truncated glycosylation. The exposed backbone is an ideal site to detect and treat tumor cells with antibodies.
AR 20.5 is a monoclonal antibody that specifically binds MUC 1 and has been shown to reduce tumor size in mouse models. Binding studies and X-ray crystallography have demonstrated that the antibody binds a conformational epitope stabilized by cancer specific glycosylation of the VNTR region. Interestingly, in the cases of other MUC1 specific antibodies the number of VNTR repeats influences the binding affinity. Based on these observations we hypothesized that increasing number of VNTR repeats has a similar conformational effect to that of cancer specific glycosylation, stabilizing the conformational epitope recognized by AR20.5. To investigate the effect of altering the number of VNTR repeats on AR20.5 binding we have constructed GST fusion proteins with 1, 3 and 5 VNTR repeats. In support of the hypothesis, indirect enzyme-linked immunosorbent assay (ELISA) employing biotinylated AR20.5 and microtitre plates coated with different GST fusions has clearly revealed that increasing the number of repeats enhances the affinity of AR20.5 for antigen.
The project is funded by Start-up funds from CSU Fresno and a research grant from Quest Pharmatech Inc.
Poster #: 163
Campus: San José State University
Poster Category: Biochemistry
Keywords: calorimetry, desolvation, thermodynamics
Project Title: Isothermal Titration Calorimetry Analysis of Desolvation Energy of Binding
Author List:
Vu, Brian; Undergraduate, Chemistry, San José State University, Presenting Author
Eggers, Daryl; Chemistry, San José State University
Abstract: In an aqueous binding reaction, the surface of two molecules will contact each other and a portion of the water that surrounds each molecule will be released into the bulk phase. The change in energy that results from this portion of water is referred to as the desolvation energy of the binding reaction. In many applications of thermodynamics, the desolvation energy is overlooked or misrepresented. However, the Eggers laboratory is testing a new approach that includes the contribution of the desolvation energy in the governing equations. This project investigates a model binding reaction between α-cyclodextrin, a truncated cylinder host, and a guest molecule, 4-nitrophenol, using an isothermal titration calorimeter (ITC). The ITC measures the heat of binding when the syringe injects a small volume of reactant, α-cyclodextrin, into the sample cell that contains the other reactant, 4-nitrophenol. Further injections continue toward saturation and create a binding curve from which the equilibrium ratio, K, may be determined. If the desolvation energy has a large energetic contribution, then our working equation anticipates that the measured K value will depend on the concentration of the two molecules of interest, even though the concentration ratio of the two reactants is held constant. To investigate the dependence of the K value with concentration, the limiting reactant concentration was varied between 1-15 mM at 25 °C. The results indicate that K decreases slightly as the reactant concentrations increases. Using our thermodynamic framework, we conclude that the desolvation energy is positive (unfavorable) for this specific binding reaction. This basic science project is important for the pharmaceutical industry and biotechnology, in general, because a better understanding of the role of water in binding reactions could lead to the design of drugs or receptors that bind to their targets with higher affinity.
Acknowledgement: This project is funded by an NIH R15 award to DKE, GM110654.
Poster #: 164
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: Post-Translational Modifications, RNA Binding Proteins, Alternative Splicing
Project Title: Role of Post-Translational Modifications in RNA Binding Domain 2 of Splicing Factor Polypyrimidine Tract Binding Protein1 Activity
Author List:
Shankar, Archana; Undergraduate, Biological Sciences, California State University, Fullerton, Presenting Author
Ramirez, Alfonso; Graduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Pina, Jeffrey; Graduate, Chemistry and Biochemistry, California State University, Fullerton
Keppetipola, Niroshika; Chemistry and Biochemistry, California State University, Fullerton
Abstract: Alternative splicing allows for the production of multiple mRNAs and hence multiple proteins from a single gene. This process is regulated in part by RNA binding proteins that bind to short sequence elements in the pre-mRNA and influence spliceosome assembly at adjacent splice sites. Spliced variants have been identified for a large number of genes associated with cancer and neurological disease underscoring the importance of splicing regulation. Post-Translational Modifications (PTM’s) in RNA binding proteins alter the protein’s splicing activity. An understanding of the molecular mechanisms of how PTM’s occur, their effect on splicing activity, and changes in cell protein composition will provide new targets for rational drug design. The Polypyrimidine Tract Binding Protein1 (PTBP1) is a well-studied splicing factor that has served as an informative model in understanding how RNA binding proteins affect spliceosome assembly. PTBP1 has four RNA binding domains connected via 3 flexible linker regions. PTBP1 functions primarily to repress inclusion of regulated cassette exons in the final mRNA transcript. PTBP1 has many PTM’s, thus serves as an ideal candidate to study how signaling pathways impact the activity of splicing regulatory proteins through PTM’s. Here, we aimed to understand the role of PTM’s of RNA binding domain 2 (RRM2) on PTBP1 splicing activity. RRM2 is post-translationally phosphorylated, ubiquitinated and acetylated at Serine/Tyrosine and Lysine residues respectively. To probe the role of each modification in PTBP1 splicing activity, we conducted an alanine scan of the modified residues. Mutants were generated by 2step PCR and cloned into the mammalian expression vector pcDNA3.1. The mutants were assayed in vivo for protein expression and splicing of a reporter minigene in HEK293T and mouse N2A cells. Western blots confirmed the mutants are well expressed. Splicing activity was assayed by isolating RNA and carrying out reverse transcription-PCR. The reaction products were analyzed by separating on a UREA-PAGE gel, visualized by a Storm860 phosphoimager and % exon excluded was quantified. Our results indicate that single mutations in RRM2 of PTBP1 do not affect its splicing activity on the assayed test exons. Several residues in the domain carry the same modification, which may lead to functional redundancy. We have constructed modification specific mutants and are currently testing for protein expression and splicing activity.
Poster #: 165
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: terpyridine, HepG2, cancer
Project Title: Toxicity of terpyridine based anti-tumor agents against HepG2 cultures
Author List:
Cannata, Angela; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Melgar, Estelli; Undergraduate, Biology, California State University, Northridge, Presenting Author
Kelson, Eric; Chemistry and Biochemistry, California State University, Northridge
Abstract: 2,2’:6’,2”-Terpyridines (Terpys) have been reported to be promising anti-tumor agents that intercalate DNA and block replication and transcription. Terpys also exhibit a promising selectivity for tumor cells that likely exploit an as yet unidentified means of cellular uptake. We have developed methodologies for conveniently making Terpys (and their corresponding ruthenium complexes) designed to intercalate DNA. The purpose of this work is to establish methodology to test substituted Terpys for anti-tumor activity and survey the possible transport mechanisms for Terpy entry into cells. After cultures of HepG2 cells were grown to near full confluence in well plates, the growth media was replaced with medium containing a range of anti-tumor agent concentrations (typically 100 µM to 2 µM). The cells were incubated for 24 hours and assayed for cellular survival via a Trypan Blue Exclusion Assay or MTT colorimetric assay. Initial experiments confirmed that Terpy was indeed toxic at 20µM for HepG2. The substituted Terpys 4’-(4-methylphenyl)terpyridine, 4’-chloroterpyridine, and 4’-(9-phenylanthryl)terpyridine showed some toxicity, but all three visibly precipitated. This limited solubility severely reduced the actual concentration, so the true toxicity requires further investigation. To guide strategies to solubilize the Terpys, the likely transport processes for Terpy itself were probed. Adding glucose to the medium increased the toxicity of Terpy to HepG2 suggesting that Terpy was entering the cells by an active transport process promoted by the additional sugar. The addition of chloroquine to the media (a general inhibitor to endocytotic processes) had no influence on Terpy toxicity indicating the toxin was not likely transported by endocytosis. Surprisingly, tetraethylammonium chloride steeply decreased the toxicity of Terpy toward HepG2 suggesting that Terpy is actively pumped into these cells through organic cation transporters. Though Terpy is a neutral molecule, it likely binds metal ions in the growth media to become an organic cation that is subsequently transported. Overall, we have used our methodology to confirm the toxicity of Terpys and identify a likely mechanism for cell uptake. Means to deliver Terpys with limited solubility are under investigation. We thank CSUPERB (Research Development Grant) and the NIH (SCORE S06 GM48680, GM063787, 8TL4GM8977-02, and 5RL5MD009603-02) for support.
Poster #: 166
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: tobacco, cancer, air quality
Project Title: Impact of Atmospheric Oxidation on the Cellular Toxicity of Cigarette Smoke
Author List:
Mann, Arjun; Undergraduate, Biology, California State University, Fresno, Presenting Author
Verhalen, Robyn; Undergraduate, Biology, California State University, Fresno
Bush, Jason; Biology, California State University, Fresno
Hasson, Alam; Chemistry, California State University, Fresno
Abstract: Various studies, especially those observing first and secondhand smoke, have confirmed the hazards of tobacco smoke on health. Tobacco smoke that exists in the atmosphere and includes mainstream and side-stream smoke has been termed secondhand smoke (SHS) or environmental tobacco smoke (ETS). When these particles linger, even in the absence of active smoking, humans may be exposed to these persistent ETS compounds—this has been termed thirdhand smoke. Thirdhand smoke is residual tobacco that comes in contact with indoor surfaces and/or can undergo chemical transformations when exposed to atmospheric species like ozone and nitrous acid.3 Recent work suggests that thirdhand smoke is an emerging environmental health risk and may be more toxic than SHS. Our aim is to quantify the effect of thirdhand smoke residue exposure on various cell lines. More specifically, the goal is to evaluate potential cytotoxic effects of smoke residue when exposed to ozone.
Thirdhand smoke filter extracts have been tested on three cell lines, A549 (human lung epithelial carcinoma), CCL-93 (Chinese hamster lung fibroblast), and TK6 (human spleen lymphoblast) using a microplate-based colorimetric WST-1 assay which measures cellular proliferation, viability, and cytotoxicity by the level of mitochondrial dehydrogenase activity. Smoke extract is applied at varying concentrations (2.5%, 2%, 1.5%, 1%, 0.75%, 0.5%, 0.25%, 0.1%, 0.05%, and 0.01%) and subsequently measured for absorbance using a plate reader at 440nm. Cell proliferation ratios were obtained relative to controls and then normalized to trial specific extracted particle masses and recorded in mg/ml. As expected, higher concentrations of thirdhand smoke residue inhibited cell proliferation most significantly. The mean IC50 values (inhibitory concentration to reduce cell response by half) for trials exposed to ozone and for standard trials (without ozone) are 0.12 ± 0.012 mg/ml and 0.15 ± 0.070 mg/ml respectively. Taken together, these results suggest that the cellular impact of the smoke residue is deleterious, particularly when exposed to ozone.
Poster #: 167
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: organophosphates, Alzheimer’s disease, cholinesterase
Project Title: Evaluating Alkyl Cholinyl Phenyl Phosphates and an Ionizable Analog as Selective Butyrylcholinesterase Inhibitors
Author List:
Dinh, Helen; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Gonzalez, Jeannette; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach
Tran, Trina; Graduate, Chemistry and Biochemistry, California State University, Long Beach
Ochoa, Jocelyn; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach
Lowe, Jacques; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach
Nakayama, Kensaku; Chemistry and Biochemistry, California State University, Long Beach
Schwans, Jason; Chemistry and Biochemistry, California State University, Long Beach
Abstract: Alzheimer’s disease (AD) is a progressive neurological disease that results in loss of cognitive function. In patients with AD, higher levels of butylrylcholinesterase activity (BChE) are observed. Acetylcholinesterase (AChE) levels are either unchanged or decreased, and the lower acetylcholine levels are suggested to contribute to AD symptoms. To reduce the depletion of acetylcholine in patients and alleviate symptoms of AD, potent and specific BChE inhibitors have been reported as potential therapeutics. We previously found that dialkyl phenyl organophosphates are potent and selective BChE inhibitors. Building on this work, we envisioned that substituting an organophosphate alkyl group with a cholinyl group may offer more potent inhibitors, as the positively charged cholinyl group may mimic the cationic choline substrate and facilitate binding interactions within the so-called choline-binding site. A series of alkyl cholinyl phenyl phosphates were synthesized and biochemically evaluated using steady-state kinetics and UV-Vis spectroscopy to determine equilibrium inhibition constants (KI values). The KI value for cholinyl ethyl phenyl phosphate was 50 μM and increasing the alkyl chain to butyl, pentyl, heptyl, and nonyl, successively led to more potent inhibitors with KI values of 10.5, 5.7, 0.6, and 0.4 μM, respectively. These values are significantly lower than the reported KI values for dialkyl phenyl phosphates. All compounds did not inhibit AChE suggesting the compounds are BChE-specific inhibitors. To dissect further the effects of charge for the cholinyl analogs, we evaluated the effect of pH on inhibition for an organophosphate bearing an ionizable amino group and a control compound lacking an ionizable group. The KI values for the ionizable analog increased as pH increased in contrast to the control compound, suggesting that the cationic group is important for inhibition. Our results show that organophosphates bearing a cholinyl group are potent and selective inhibitors of BChE, suggest the positively charged cholinyl group is important for inhibitor binding, and provide the foundation for using cholinyl organophosphates as BChE specific inhibitors.
This project was supported by the National Institute of General Medical Sciences, National Institutes of Health (NIH) MARC U*STAR program under award number T34GM008074. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Poster #: 168
Campus: CSU San Marcos
Poster Category: Biochemistry
Keywords: Hsp60, protein stability, fluorescence
Project Title: Denaturation Curve of HSP60 from Helicobacter pylori: Effect of GTP
Author List:
Guadalupe, Karina; Undergraduate, Chemistry and Biochemistry, California State University San Marcos, Presenting Author
Mendoza, Jose; Chemistry and Biochemistry, California State University San Marcos
Abstract: Background: Helicobacter pylori, the cause of most gastritis cases, thrives in the highly acidic pH of the human stomach. The heat shock protein Hsp60 is one of the most abundant proteins observed in H. pylori. Since the structure and sequence of Hsp60 are similar to those of the chaperonin from E. coli, GroEL, it is hypothesized that their functions are similar as well. However, unlike GroEL which binds to ATP, Hsp60 from H. pylori binds to GTP. In a previous study, it was shown that the binding of GTP caused alpha-crystallin, a protein in the eye lenses that functions as a molecular chaperone, to be less stable. Here, we investigated the effect of GTP on the chemical stability of H. pylori Hsp60.
Methodology: The wild type Hsp60 does not contain any tryptophans. Therefore, a mutant of Hsp60 containing a tryptophan replacement at residue 202 (Y202W) was purified and used for fluorescence studies. Samples of 1 mL aliquots were prepared (final concentrations in aliquots were 50 mM Tris-HCl pH 7.5, 1 uM Y202W, 0.25 mM GTP, and with varying urea concentrations (0-6 M). Blanks were subtracted from samples. All aliquots were incubated for 24 h before their fluorescence was measured between 310 nm and 370 nm using a Fluoromax-3 spectrophotometer. Samples were excited at 280 nm.
Results and Discussion: The native state of the Hsp60 mutant displayed a maximum fluorescence intensity at 331 nm. The Hsp60 mutant appeared to be completely denatured after its incubation with 4 M urea or higher concentrations. The denatured state of the Hsp60 mutant displayed a maximum fluorescence intensity at 353 nm. Fluorescence spectroscopy was then used to determine whether the binding of GTP affected the stability against urea denaturation. Our results show that Hsp60 has a relatively low stability against urea denaturation (C1/2 = 2.75 M) and that the binding of GTP had a small destabilizing effect. A C1/2 of 2.50 M was observed for the Hsp60 mutant in the presence of GTP.
Acknowledgment: This work was supported in part by the Office of Training, Research and Education in the Sciences at CSUSM.
Poster #: 169
Campus: CSU East Bay
Poster Category: Biochemistry
Keywords: RNA, Brome Mosaic Virus, Fluorescence
Project Title: An RNA tri-loop can drive protein-RNA interactions: Studies on the binding characteristics of the N-terminal region of a viral coat protein on its RNA
Author List:
Smith, Edward; Graduate, Chemistry & Biochemistry, California State University, East Bay, Presenting Author
Court, Gabrielle; Undergraduate, Chemistry & Biochemistry, California State University, East Bay, Presenting Author
Kim, Chul-Hyun; Chemistry & Biochemistry, California State University, East Bay
Abstract: Brome Mosaic Virsu (BMV) is an RNA virus that infects granary plants. Its RNA replication mechanism has been extensively studied because of its economic significance as well as its importance as a model system for clinically important viruses such as Hepatitis C virus. A loop consisting of three nucleotides (5’AUA3’) was found at the 5’end of BMV genomic RNA. It serves as a key recognition motif for the specific binding of an RNA polymerase on BMV RNA. Its solution structure showed that the adenine base on the 5’ side of the triloop is crucial in forming a slanted surface for enzymatic recognition. Successful RNA replication in BMV also requires a regulatory function conducted by its coat protein (CP), which involves the specific binding between the N-terminal region of CP and the RNA. Here, we hypothesized that the same 5’AUA3 triloop also serves as a recognition motif for the CP-RNA interactions. In this study, we have investigated the binding characteristics between 5’AUA3’ and the N-terminal region of CP using fluorescence quenching and anisotropy as well as high-resolution NMR. We prepared a peptide (CPNT) that mimicked the N-terminal region of CP and three RNA sequences that resemble the wild-type triloop (5’AUA3’), its mutant with a replacement of A on the 5’ side with G (5’GUA3’), and an RNA with a random conformation (poly-U RNA). Our NMR data showed that CPNT itself is not well structured, yet undergoes significant conformational changes in the presence of the RNA, evidenced by the 2D HSQC spectra. Our fluorescence data showed that CPNT binds 5’AUA3’ with significantly higher affinity than 5’GUA3’, suggesting that 5’AUA3’ triloop motif indeed serves as a crucial recognition motif for the binding of CPNT. CPNT minimally bound to poly-U, confirming that the specific binding of CPNT-RNA is mostly driven by structure-based interactions rather than general electrostatic interactions. From the anisotropy measurement, the dissociation constant of CPNT-5’AUA3’ interactions was estimated to be about 50 μM, which indicates relatively weak binding. This suggests that CPNT uses the multiple parts of the genomic RNA molecule as its recognition motifs in its binding, which is consistent with the findings of recent studies. Our results confirm the interesting role of this tiny loop (5’AUA3’) as a universal recognition motif for important protein-RNA interactions in the RNA replication process of BMV.
Poster #: 170
Campus: Sonoma State University
Poster Category: Biochemistry
Keywords: fuel cells, hydrogenase , bioinoganic chemistry
Project Title: Mechanistic Investigation of Photochemical Products from Iron-Iron Hydrogenase Model Compounds
Author List:
Nelson , Anne ; Graduate, Chemistry , Sonoma State University, Presenting Author
De La Torre, Patricia; Undergraduate, Chemistry , Sonoma State University, Presenting Author
Works, Carmen ; Chemistry, Sonoma State University
Abstract: The Fe-Fe hydrogenase enzyme reversibly catalyzes the oxidation of molecular hydrogen with incomparable efficiency. Hydrogenases are found in bacteria to help them metabolize hydrogen, and carbon monoxide (CO) is a competitive inhibitor for the substrate. CO inhibition is photochemically reversible. This enzyme class has gained significant attention because it represents renewable biohydrogen, as well as a platform for hydrogen fuel cell studies and development. Crystal structures isolated from anaerobic bacteria have incited interest in structural and functional small models of the enzyme active site. These structurally simplistic models are a powerful means to study the active site chemistry of hydrogenases. Structural and functional models of the general formula (μ-Rdt)[Fe2L6] are showing great promise as iron-based catalysts for hydrogen generation and activation. These models offer an attractive replacement for costly platinum analogs, which are the basis of current hydrogen fuel cells. Additionally, the models are photochemically active, which we hypothesize is important for catalytic activity. Our research is focused on investigating the wavelength dependent role of light, which is required to drive the hydrogen evolution reaction in [FeFe]-hydrogenase models. Additionally, solvent is examined because of its varying impacts on the photochemistry of these models.
Previously we have shown that limited photolysis of (μ-pdt)[Fe2(CO)6] [1] using 365 nm light labilizes a CO ligand and hypothesized that photolysis of (μ-pdt)[Fe2(CO)4(PMe3)2] [2] and (μ-pdt)[HFe2(CO)4(PMe3)2]+ [2H]+ would similarly remove a CO ligand, thereby opening a coordination site to bind and possibly oxidize H2. Results from FTIR and NMR indicate unique chemistry for each derivative in the presence of dissolved N2, CO, and H2. Data will be presented to support photochemical induced CO loss which leads to the formation of a solvento species in coordinating solvents, including coordination of a third phosphine ligand under some conditions. While it is clear that loss of the hydridic proton is not the primary photolysis product of [2H]+, thermal regeneration of the neutral species in solution contributes to observed spectral changes as indicated by post-photolysis growth of peaks seen in the IR spectrum of [2]. From these studies, we conclude that photolysis using 365 nm light specifically releases a CO ligand, and generates a model of the active biological catalyst.
Poster #: 171
Campus: CSU Stanislaus
Poster Category: Biochemistry
Keywords: cigarette, radical, DNA
Project Title: Biochemical Characterization of the Cigarette Tar Radical in Third Hand Smoke
Author List:
Skochko, Alexander ; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author, Nagel Award Finalist
Lopez, Chris ; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Russell, Scott; Chemistry, California State University, Stanislaus
Stone, Koni ; Chemistry, California State University, Stanislaus
Abstract: Exposure to cigarette smoke has been linked to a higher risk of cancer for both smokers and non smokers. Recently, the residue that is deposited on solid surfaces, also known as “third hand smoke”, has been implicated as a health threat. Aqueous extracts of mainstream and sidestream smoke from cigarettes contain a stable polyphenolic radical polymer. These polymers bind to DNA and induce single strand nicks in DNA that are not easily repaired. Environmental residue from cigarette smoking, also known as “third hand smoke,” may contain this tar radical. The goal of the present research is to study the environmental residue by purifying the tar radical from side stream smoke, which comes off of the tip of the cigarette, and compare it to the tar from third hand smoke. An apparatus was constructed to collect these types of cigarette tar on cellulose filters. Then, the aqueous extracts from each type of cigarette tar were purified by G25 Sephadex chromatography. The fractions were analyzed by UV spectroscopy and then tested for DNA nicking. A literature procedure was modified to develop a high throughput agarose gel electrophoresis assay for DNA nicking. Super-coiled, double stranded plasmid DNA (pUC18) unwinds when a single strand is nicked. This produces two bands in the agarose gel: nicked and intact supercoiled DNA. The gels were stained, scanned, and quantitated using Image-J software. DNA nicking was detected and quantitated when DNA was incubated with solutions of side-stream or third hand smoke. DNA that was incubated with buffer alone exhibits 0-4.1% DNA nicking. When DNA is incubated with either side stream or environmental residue there is 8-35% DNA nicking. There is a similar dose response for both types of smoke. Indole-3-carbinol (I3C) is a compound that is present in broccoli (up to 500 ppm), it has been shown to block DNA damage that is caused by radicals. We tested I3C with tar extracts in our DNA nicking assay. Our preliminary results are confounding and do not support the previously reported results. Also, when only I3C is incubated with pUC18 the supercoiled DNA band is broadened and shifted to a lesser mobility. The purification, UV spectra and DNA nicking results support our conclusion that environmental smoke residue can cause DNA damage and it is chemically similar to the tar radical that is present in side stream (and main stream) cigarette smoke.
Poster #: 172
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Bcl-2, LDH, oxamate
Project Title: Study of the effect of Bcl-2 overexpression on oxamate-induced cytotoxicity in prolymphocytes
Author List:
Chong, Hooi; Undergraduate, Biology, California State University, Fresno, Presenting Author
Kasnakjian, Lucineh; Undergraduate, Biology, California State University, Fresno, Presenting Author
Olino, Patricia; Undergraduate, Nursing, California State University, Fresno
Bader, Garrett; Undergraduate, Chemistry, California State University, Fresno
Ly, Benjamin; Undergraduate, Chemistry, California State University, Fresno
Mahmood, Bushra; Graduate, Chemistry, California State University, Fresno
Dejean, Laurent; Chemistry, California State University, Fresno
Abstract: Research on apoptosis and control of gene expression has created various types of treatments to benefit the cancer community. Apoptosis is a programmed cell death which rids the body of potentially toxic cells. This death program is often inhibited in cancerous blood cells. In mammalian cells, glucose acts as a major fuel source to generate energy. Glucose breaks down into pyruvate via glycolysis. In normal aerobic cells pyruvate enters the mitochondria where it is oxidized by the Krebs Cycle to generate ATP. However, in cancerous cells, it is believed that an increased portion of this pyruvate generated by glycolysis is used for lactic fermentation; a process requiring the enzyme called lactate dehydrogenase (LDH). In actuality, there had been several studies showing that LDH inhibitors such as Oxamate can impact more severely growth and survival of cancer cells vs. normal cells.
The Bcl-2 family of proteins are well-known regulator of apoptosis; and an increase of the expression levels of their anti-apoptotic members such as Bcl-2 have been positively associated with cancer transformation for more than two decades. We recently observed that, in the murine prolymphocytes FL5.12 cell line, the over-expression of Bcl-2 led to an increase in lactate production. Increase of lactate production indicates an up-regulation of fermentative metabolism. This process potentially mirrored how cancer cells use lactate fermentation to produce energy. We therefore studied the dose response relationship of the LDH inhibitor Oxamate on the survival of Bcl-2-overexpressing vs. Parental FL5.12 cells. Surprisingly, we observed than higher doses of Oxamate were required to kill Bcl-2-overexpressing vs. Parental cells. This result suggests that the fermentative component of glucose metabolism is less critical for survival in Bcl-2 overexpressors; and consequently that Bcl-2 over-abundance does not a trigger a Warburg-type phenotype to pro-lymphocytes. Future studies involve the testing of the same cell line over-expressing other Bcl-2-family type proto-oncogene such as Bcl-xL.
Poster #: 173
Campus: San José State University
Poster Category: Biochemistry
Keywords: Directed Evolution, Cytochrome P450, light-driven
Project Title: Using a Directed Evolution Approach for the Optimization of Light-Driven P450 Biocatalysts
Author List:
Li, Jennifer; Undergraduate, Chemistry, San José State University, Presenting Author
Tang, Lawrence; Undergraduate, Chemistry, San José State University
Kato, Mallory; Staff, Chemistry, San José State University
Cheruzel, Lionel; Chemistry, San José State University
Abstract: The demand for green and sustainable synthesis of various fine chemicals and pharmaceuticals has rendered cytochrome P450 enzymes of biotechnological interest due to their great synthetic potential. These heme thiolate enzymes catalyze the hydroxylation of unactivated C-H bonds with high regio- and stereoselectivity. Molecular dioxygen is used as the oxygen atom source and the two electrons required for the reactions to occur are delivered to the active site using reductase, an electron transfer enzyme that requires the cofactor NADPH.
In an effort to eliminate the use of the electron providing reductase and NADPH cofactor, our laboratory has established a light-driven system capable of harnessing the synthetic potential of P450 enzymes upon visible light excitation. Our prototype hybrid enzyme consisted of a mutated heme domain of the P450 BM3 enzyme with a covalently attached Ru(II)-diimine photosensitizer. This efficient hybrid enzyme showed high photocatalytic activity in the hydroxylation of various natural substrates.
Recently, we have sought to increase the photocatalytic activity of our hybrid enzyme system by expanding our mutant library through a directed evolution approach. The success of this approach relies on the rapid screening of a large number of randomly generated variants. Herein we report the implementation of a throughput screening of light-driven hybrid enzyme mutants in our laboratory. Using error-prone PCR, we generated random mutations in the gene coding for the heme domain of our efficient mutant with a mutation rate resulting in 1-3 amino acid changes per construct. The resulting mutants were expressed and the covalent attachment of the photosensitizer was adapted to a 96-well plate format. The photocatalytic activity of the hybrid enzyme mutants was rapidly determined using our recently reported colorimetric assay, which utilizes various nitrophenoxy-based compounds as chromogenic substrates. Early screening of the mutants has shown an increase in activity of greater than 50%, demonstrating the potential of this approach for the optimization of our light-driven hybrid enzymes.
Poster #: 174
Campus: Sonoma State University
Poster Category: Biochemistry
Keywords: Phosphatase, Oxidation, Cell Signaling
Project Title: Analysis of Protein Tyrosine Phosphatases in Varying Redox Conditions
Author List:
Grunberger, Jason; Undergraduate, Biology, Sonoma State University, Presenting Author
Arellano, Fernando; Undergraduate, Biology, Sonoma State University, Presenting Author
Fukuto, Jon; Chemistry, Sonoma State University
Lin, Joseph; Biology, Sonoma State University
Abstract: Tyrosine phosphorylation and dephosphorylation of proteins comprise key regulatory events in many signal transduction pathways. Tyrosine Kinases attach phosphate onto specific tyrosine residues whereas Protein Tyrosine Phosphatases (PTPs) catalyze the hydrolysis of phosphate from phosphorylated tyrosine residues. These PTPs therefore play an important role in the regulation of signal transduction. All PTPs contain an active site cysteine required for their catalytic activity. Importantly, the cysteine must be in the reduced state. However, it is clear that the cysteine can also be regulated via reversible oxidation. In this study, we focus on thiol-mediated reactivation of three different oxidized protein tyrosine phosphates: SHP1, CD45, and CD148. It is important to note that all three PTPs have been shown to act on the same substrate.
To begin, we cloned the phosphatase domains from the respective PTPs and inserted a poly-histidine tag. PTPs were then purified by affinity chromatography using ni-NTA resin. PTPs were oxidized with H2O2 to render a baseline inactive state of our enzymes. Then, varying concentrations of Glutathione and Dithiothreitol were used to reduce the active site cysteine to recover catalytic activity. We observed recovery of phosphatase activity in all three PTPs to differing degrees depending on the PTP demonstrating that SHP1, CD45 and CD148 have varying response to reductive reactivation. Future research will focus on a physiological explanation behind these varied responses to redox regulation involved with cell signaling.
Poster #: 175
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: copper absorption intestine, ,
Project Title: Copper uptake by intestinal cells across the brush border occurs through more than one transporter, as demonstrated in the Caco2 cell culture model
Author List:
Wertz, Aleksander; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Ricarte, Adrian; Graduate, Chemistry and Biochemistry, California State University, Fullerton
Linder, Maria C; Chemistry and Biochemistry, California State University, Fullerton
Abstract: The only currently identified copper-specific uptake transporter for Cu in mammalian cells is CTR1 (Cu transporter 1), which absorbs Cu(I) ions. However, for uptake of dietary Cu by intestinal cells, there are many controversies, including whether CTR1 even occurs in the apical (brush border) of the cells; whether DMT1 (divalent metal transporter 1), the main uptake transporter for Fe and Mn, might also be involved – particularly in iron deficiency; and whether a Cl-dependent mechanism may also participate. The studies reported here were initiated to resolve these questions, using the human Caco2 intestinal model and 67Cu radiotracer to measure Cu uptake rates.
Caco2 cell monolayers were grown in DMEM/10% fetal bovine serum on filters coated with rat tail collagen in Transwells until resistance >200 Ohms, indicating tight junctions. Uptake of 67Cu-labeled Cu(I)-nitrilotriacetate (5 uM) with 1 mM ascorbate, applied to the apical (brush border) side, was linear over 60 min. Uptake rates were calculated based on radioactivity in cells and basolateral fluid after 60 min, and the protein content of cell lysates (Bradford assay) to give rates in pmol/min/mg cell protein. Effects of Cu and Fe status were determined by 24 h preincubation with/without 5 uM extra Cu or Fe (to induce metal enrichment) or specific Cu and Fe chelators (TETA and DFO, respectively) to induce deficiency. Silver ions were used to inhibit CTR1; sulfate was substituted to measure Cl-dependent uptake.
Ag(I) caused a small but statistically significant inhibition of Cu(I) uptake (averaging 20%). In contrast, substitution of 150 mM sulfate for chloride markedly reduced uptake (averaging about 70%). In the presence of sulfate plus silver, rates of uptake were still about 15% of the total rate without these inhibitors suggesting an additional transport mechanism was present. Pretreatment with extra Cu or Fe failed to change uptake rates; however, copper deficiency decreased rates about 18%, while Fe deficiency had no effect. Knockout of CTR1 and DMT1 was initiated with CRISPR-Cas9 technology to further assess contributions of these transporters. Cells that survived treatment are currently being assessed for knockout.
We conclude that at least in this intestinal model, chloride-dependent transport is the major means by which Cu(I) ions are taken up across the brush border, and that CTR1 is only a minor contributor to this process.
Poster #: 176
Campus: CSU Los Angeles
Poster Category: Biochemistry
Keywords: protein, calcium carbonate, crystallization
Project Title: Effects of ice-binding proteins from cold-adapted insect Tenebrio molitor on calcite crystallization
Author List:
Kishishita, Audrey; Undergraduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Lugo, Joshua; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Wen, Xin ; Chemistry and Biochemistry, California State University, Los Angeles
Abstract: The formation of sparingly soluble and insoluble inorganic salts (i.e., scale deposits) is a major problem an industrial and domestic setting. To control scale deposits, chemical scale inhibitors are commonly used. Commercial antiscaling agents include polyelectrolytes that dissociate phosphonates, carboxylates, and sulfonates anionic groups. However, it is imperative to identify highly efficient polymeric inhibitors and environmentally friendly antiscalants, in particular, to replace phosphonate inhibitors due to their environmental risks. Certain polypeptides with charged groups have been extracted from organisms and found to efficiently control the nucleation and crystallization of minerals. Their structures are attractive models for better understanding the inhibitor-mineral interactions and designing next generation antiscalants. Ice-binding proteins or antifreeze proteins (AFPs) from cold-adapted organisms (e.g., fish, insects, and plants) can bind to specific ice surfaces, thereby inhibiting the nucleation and crystallization of ice. AFPs cam also control the crystallization of some non-ice like compounds by interacting with the crystalline surfaces of these compounds. We correlate the charge and molecular properties of the polyelectrolytes with their efficiencies in inhibiting the scale crystal formation. A beetle AFP from Tenebrio molitor (TmAFP) having regular spaced charged residues on its surfaces is prepared and studied here. Calcium carbonate (CaCO3), a common scale deposit, is a scalent of interest in this study. We investigate the effects of TmAFP on the formation of CaCO3. Our results show that the presence of TmAFP inhibits the formation of CaCO3 resulting much fewer CaCO3 crystals. The achieved CaCO3 crystals in the absence and presence of TmAFP are both characterized to be calcite using Fourier transform infrared (FTIR) spectroscopy. The results suggest that the presence of TmAFP does not change the polymorph of CaCO3. By analyzing the charged residues on the surfaces of TmAFP and calcite surfaces, we propose that TmAFP may affect the formation of calcite via adsorption to the crystalline surfaces of CaCO3. This study provides better understanding for scale control as well as new designs for green antiscalants.
Poster #: 177
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: metal transport, fluorescence microscopy, neurodegenerative disease
Project Title: Characterization of ionic zinc flux in cells expressing the transmembrane (TMEM)-163 protein
Author List:
Rivas, Tiffany; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Shoemaker, Ryan; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Cantrell, Quinlan; Undergraduate, Biological Science, California State University, Fullerton
Cuajungco, Math; Biological Science, California State University, Fullerton
Abstract: An elevation of chelatable zinc, particularly in lysosomes, is observed in cells affected by Mucolipidosis type IV (MLIV) disease compared to normal cells. MLIV is caused by the functional loss of Transient Receptor Potential Mucolipin-1 (TRPML1) ion channel. We recently implicated TMEM163 as a possible contributing factor behind the zinc buildup in MLIV cells based on its interaction with TRPML1 and its putative role as a zinc transporter. We proposed that TMEM163 is an protein transporter that facilitates zinc influx in normal cells. To test our hypothesis, we cloned TMEM163 and several known zinc transporters (ZNTs and ZIPs) into the pBI-CMV dual-expression vector, and heterologously expressed the constructs in human embryonic kidney (HEK)-293 cells. Spectrofluorometric end-point assay using the zinc-specific Fluozin-3 dye showed that cells over-expressing TMEM163 produce intracellular zinc accumulation when transiently exposed to zinc chloride (ZnCl2, 100 uM). Interestingly, cells co-expressing TMEM163 and ZNT4, but not TMEM163 and other ZNTs, have higher intracellular zinc levels at specific time points tested. To further confirm zinc influx in TMEM163-expressing cells, we transfected HEK-293 cells with TMEM163 tagged with a red fluorescent protein and performed live cell imaging using Fluozin-3 dye to visualize intracellular zinc flux. We found that cells that express TMEM163 accumulate intracellular zinc relatively faster (~10 minutes) upon exposure to ZnCl2 (50 uM) compared with cells that do not express TMEM163 (~50 minutes). Quantitative analysis of the Fluozin-3 fluorescence validates the live cell imaging data. Overall, these results suggest that TMEM163 may belong to a new class of zinc influx transporter that is structurally unique to ZNTs or ZIPs. Future studies will further address the kinetics of zinc transport mediated by TMEM163. Overall, our observations provide new research opportunities to further understand the involvement of zinc in the underlying neuropathology seen in MLIV disease.
This work was funded by the CSUPERB Research Development Grant Program 2016.
Poster #: 178
Campus: CSU East Bay
Poster Category: Biochemistry
Keywords: Tritonia diomedea, enzyme, detoxification
Project Title: Characterization of the detoxification enzyme glutathione-S-transferase from the marine gastropod Tritonia diomedea
Author List:
Cooney, Samuel; Graduate, Chemistry and Biochemistry, California State University, East Bay, Presenting Author
Huang, Shuang; Graduate, Department of Chemistry and Biochemistry, California State University, East Bay
Murray, James; Biology, California State University, East Bay
Sommerhalter, Monika; Chemistry and Biochemistry, California State University, East Bay
Abstract: The marine gastropod Tritonia diomedea (a.k.a. T. tetraquetra) is a popular invertebrate model in behavioral neurobiology. This gastropod is able to feed on a poisonous sea pen, Ptilosarcus gurneyi. To gain more insight into the gastropod’s tolerance we characterized its detoxification enzyme glutathione-S-transferase (GST). Marine organisms use up to five different GST isoforms to defend themselves against toxins.
T. diomedea were collected close to Tofino (British Columbia, Canada) and Dash Point (Washington, United States). The gastropods were dissected to prepare tissue extracts from digestive glands. GST was purified via affinity chromatography using agarose with immobilized glutathione as bait molecule. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a single GST isoform. Enzyme activity was measured with the substrates glutathione and 1-chloro-2,4-dinitrobenzene(CDNB). Protein content was determined via Bradford assays.
Specific GST activity values for Dash Point samples were 4-times greater than for Tofino samples for a sample set of eight gastropods per collection site. Genetic differences in population or environmental factors, e.g. water quality, can result in an increase of GST activity. Our enzyme kinetic data indicates that T. diomedea possess a novel GST isoform. Substrate inhibition, as observed for our samples above 2.5 mM CDNB, is rarely reported for marine GSTs. Also the Michaelis-Menten constant for glutathione fell above 1 mM whereas most other marine invertebrate GSTs show values below 1 mM. For further characterization we aim to obtain the amino acid sequence via mass spectrometry analysis. Since this method requires a large protein quantity we optimized our purification method. Several affinity chromatography variations, including batch chromatography, gravity flow columns, spin columns, and pre-packed columns for fast protein liquid chromatography (FPLC), were employed. We found that the spin columns provided the largest yield and were the most expedient method. For 3.5 g digestive gland tissue we obtained a final protein yield of 4 mg.
This project was funded by a Faculty Support Grant for Collaborative Research from CSU East Bay.
Poster #: 179
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: antibodies, environmental pollutants, structure
Project Title: Physical properties explored in the process of co-crystallization of camelid variable heavy chain domain antibodies used to detect harmful chemicals
Author List:
White, Brandy; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Bever, Candace; UC Davis
Gee, Shirley; UC Davis
Hammock, Bruce; UC Davis
Brooks, Cory; Chemistry, California State University, Fresno
Abstract: Brominated chemical flame retardants (BCFRs) have been used extensively in recent decades in furniture, plastics, wiring, and electronics because they were thought to prevent fires in homes and workplaces. However, there is growing concern of these BCFRs in living spaces as there is evidence of bioaccumulation in human blood, breast milk, and body fat, and while they mimic the endocrine system in rat models, they also have high potential of being carcinogenic. While BCFRs have recently been placed on the California Biomonitoring Program and have been banned by the EPA, these BCFRs persist in the environment. A detection method has been developed using a camelid variable heavy chain domain antibody (VHH)-based method of two of these BCFRs: 2,2’,4,4’-tetrabrominated diphenyl ether (BDE47) and Tetrabromobisphenol A (TBBPA). However, it is not well understood how these detection methods work and it is difficult to improve on the methods without this understanding. Camelid VHH have many advantages compared to conventional monoclonal antibodies including high stability and ease of recombinant production. Although it is well established how VHH interact with proteins, it is less well understood how these antibodies interact with small molecules and haptens. There are currently four known binding modes available for VHH to interact with small molecules including: combining VHH’s three CDR loops, domain swapping utilizing a 2:1 binding stoichiometry, and tunneling under the CDR1 loop. Using VHH specific for PBFRs as a model, we aim to examine VHH-small molecule interactions. We have successfully sequenced and overexpressed three anti-BDE47 and one anti-TBBPA proteins, as visualized with SDS-PAGE and size-exclusion chromatography. Crystallization trials of the four proteins have yielded preliminary crystals and our current focus is on crystal optimization. Additionally, physical properties of these VHH have been observed, including cold and light sensitivity. We anticipate this study to allow visualization of VHH-small molecule binding and that this will contribute to the bioengineering of future VHH-detection methods.
Funding: Howell-CSUPERB Research Scholars Award, CSUF-ASI (FSSRA), CSUF-rGrant, CSUPERB Student Travel Grant, Howard P Kubo MD Award, Helen Gigliotti Biochemistry Scholarship, Carl E Levin Science & Math Scholarship
Poster #: 180
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: mutations, heat-shock, cell survival
Project Title: Functional characterization of natural variants found on the major stress inducible 70kDa heat shock gene in humans
Author List:
Oliverio, Ryan; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Nikolaidis, Nikolas; Biological Science, California State University, Fullerton
Abstract: The cellular stress response system is responsible for maintaining cellular homeostasis during periods of stress caused by environmental insults including pathogens and disease. Molecular chaperones, and, in particular the 70-kDa heat shock proteins (Hsp70s) are key orchestrators of the stress response system. Therefore, we hypothesized that natural occurring variations in the sequence of Hsp70s will affect cellular and subsequently species adaptation. To investigate the relationship between adaptation, disease susceptibility, and Hsp70s’ variation we tested whether natural single nucleotide polymorphisms found on HSPA1A, the major stress inducible Hsp70 gene in humans, alter its function. Specifically, the wild-type human HSPA1A sequence was subcloned into mammalian expression vectors, and the mutated gene variants were generated using site-directed mutagenesis. We then determined whether any of these mutations affected the intracellular localization of HSPA1A. These experiments were performed by using GFP-tagged HSPA1A and fluorescent dyes to stain the nucleus, mitochondria, and lysosomes, and visualized using confocal microscopy. These assays revealed that the mutants and the WT protein had similar subcellular localization. Furthermore, we determined whether the mutations affected the ability of HSPA1A to prevent cell death by inhibiting the formation of protein aggregates caused by poly-glutamine carrying huntingtin proteins. This assay determined that one of the mutations caused increased cell death as compared to the WT, suggesting that it may alter HSPA1A’s ability to prevent protein aggregation. We also determined whether these mutations affect the ability of HSPA1A to refold heat-denatured luciferase. These assays revealed that only one mutation resulted in significantly lower levels of refolded enzyme, suggesting a putative loss-of-chaperone function. Lastly, live-dead assays revealed that human cell lines carrying two of these mutations had significantly less resistance to heat and ethanol stress than cells expressing the WT protein. Given that these natural variants are found at a very low frequency in humans we suspect that the observed functional differences alter the ability of cells, and the individuals carrying them, to cope with stress.
Poster #: 181
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: ER stress, Cell survival, Akt pathway
Project Title: Gɑ-Interacting Vesicle Associated Protein Mediates Cell Survival during Endoplasmic Reticulum Stress.
Author List:
Nguyen, Peter; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Rodriguez, Yoanna; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Luong, May; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach
Bhandari, Deepali; Chemistry and Biochemistry, California State University, Long Beach
Abstract: Endoplasmic Reticulum (ER) is a site of many important cellular functions including proper protein folding, post-translational modifications and maintaining Ca2+ homeostasis in virtually every mammalian cell. Perturbations in these functions lead to activation of a cellular stress response known as the unfolded protein response (UPR). Once activated, its main function is to re-establish normal ER function by decreasing general translation and increasing production of chaperones as well as ER-associated degradation of unfolded proteins. Initially, cells try to maintain homeostasis by promoting cyto-protective signaling. However, if normal ER function cannot be restored in a timely manner, cells commit to apoptosis. Cancer cells are susceptible to ER stress due to intrinsic and extrinsic factors such as elevated glucose metabolism and hypoxia. However, unlike normal cells, cancer cells manage to survive and recover from high levels of ER stress. Here, we show that GIV (Gɑ-Interacting Vesicle associated protein), a multimodular signaling protein, promotes cell survival during ER stress via activation of the Akt pathway. HeLa cells (Cervical cancer) treated with various ER stressors (tunicamycin, dithioreitol and thapsigargin) activate the Akt pathway, as determined by western blotting for the active form, phospho-Akt (pAkt). This activation is significantly diminished upon shRNA-mediated depletion of GIV. Furthermore, GIV-depleted cells show a significant decrease in cell survival during ER stress, as measured by a cell viability assay. Interestingly, stable expression of GIV’s C-terminal region alone was able to rescue the pAkt levels in GIV-depleted cells. Taken together, these findings suggest that GIV may play an important role in helping cancer cells survive ER stress. Since GIV’s C-terminus contains the Guanine nucleotide exchange factor (GEF) activity, our current and future studies are focused on using selected point mutants to determine if the GEF activity is required for GIV’s role in promoting cyto-protective signals during ER stress.
This project is supported by the CSUPERB New Investigator Grant GF00631143; the NIH BUILD Research Stimulation Grant UL1MD009601; and the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R25GM071638
Poster #: 182
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: Methylobacterium extorquens, Methanopterin biosynthesis, Enzyme assay
Project Title: Insight into the function of ORF20 from Methylobacterium extorquens AM1 in the biosynthesis pathway of tetrahydromethanopterin
Author List:
Lu, Ryan; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Gonzalez, Stephen; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Zada, Suzan; Undergraduate
Rasche, Madeline; Chemistry and Biochemistry, California State University, Fullerton
Abstract: Methane-producing microorganisms in cattle contribute to release of the greenhouse gas methane, which contributes significantly to global climate change. The coenzyme tetrahydromethanopterin (H4MPT) is a one-carbon carrier during methanogenesis, and inhibition of H4MPT biosynthesis could reduce the amount of methane emissions from cattle. The ORF20 protein from Methylobacterium extorquens AM1 has been proposed to catalyze the second of nine steps in the biosynthesis of the H4MPT side chain. The goal of the current work is to develop a biochemical assay to test this hypothesis by combining ORF20 with 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPPP) and 4-β-D ribofuranosylaminobenzene-5’-phosphate (RFAP) to determine if 6-hydroxymethyl-7,8-dihydropterin-RFAP and pyrophosphate are produced. The substrate RFAP was synthesized by combining para-aminobenzoic acid and phosphoribosyl pyrophosphate with partially purified RFAP synthase and quantifying the product using the Bratton-Marshall assay. The second substrate DHPPP was produced by Pd-catalyzed hydrogenation of 6-hydroxymethyl-pterin pyrophosphate (HMPPP), and synthesis of DHPPP was analyzed using changes in the UV-visible spectra. Phosphate released in the presence of inorganic pyrophosphatase from synthesis of 6-hydroxymethyl-7,8-dihydropterin-RFAP was quantified using a Malachite green (MG) assay. The results showed successful synthesis of RFAP with an initial concentration of 68 µM and a phosphate concentration of about 82 µM. After running an additional column to separate RFAP from phosphates, the RFAP concentration was about 65 µM, and the phosphate concentration was reduced to 7.8 µM, which was acceptable for use in proposed ORF20 assay. Comparison between the UV spectra of HMPPP and DHPPP showed an absorbance increase of 0.25 units at 230 nm, along with a decrease of 0.1 units at both 280 nm and 330 nm, consistent with successful production of DHPPP. When RFAP, DHPPP, ORF20, and inorganic pyrophosphatase were combined, the MG assay showed an increase in phosphate concentration of at least 10 µM over a 30-min time period. No increase was seen in the corresponding control assay using heat-inactivated ORF20. Based on the results of the ORF20-MG assay, this work establishes a new biochemical assay to kinetically characterize ORF20 as a novel enzyme catalyzing the second reaction of H4MPT biosynthesis.
Funding sources: CIRM Bridges to Stem Cell Research Program, NSF grant CHE-1508801, CSUPERB.
Poster #: 183
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: DNA Repair, cloning, Fluorescence microscopy
Project Title: Construction of Yeast Strains to Determine Over-hanging Flap Length Triggering Saw1-Dependent Rad1-Rad10 Recruitment During Single Strand Annealing
Author List:
Camberos, Juan; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Odango, Rowen; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Fischhaber, Paula; Chemistry and Biochemistry, California State University, Northridge
Abstract: Unrepaired DNA damage can compromise health and lead to cancer and aging. Elucidating DNA repair mechanisms including double strand break (DSB) repair, can reveal new treatment strategies. One such DSB repair pathway is single strand annealing (SSA), which occurs when a DSB arises between DNA repeats. DSB ends are resected in a 5′ to 3′ direction to expose homologous DNA sequences that anneal together. The non-homologous sequences originally located between the repeats form 3′ overhanging DNA flaps that are cleaved by an endonuclease complex which, in the yeast, S. cerevisiae, is Rad1-Rad10. Prior research shows that Saw1 recruits Rad1-Rad10 to SSA repair sites, however, recent studies in vivo suggest Saw1-independent recruitment of Rad1-Rad10 occurs when overhanging flaps are short (~10 bases) but in vitro work suggests that slightly longer flaps (~20 bases) may engage Saw1.
The ultimate goal of this study is to determine the precise flap length triggering Saw1-dependent Rad1-Rad10 recruitment in SSA. Toward achieving this goal, we prepared yeast strains containing a fluorescently labeled DSB site (DSB-RFP) flanked by DNA repeats spaced at various intervals, and a fluorescently labeled Rad10 gene (Rad10-YFP). Upon DSB induction, the strains will be monitored by fluorescence microscopy for recruitment of Rad10-YFP to DSB-RFP sites. Experimental results from pairs of strains either containing wild-type SAW1 or deleted of SAW1 will be compared to determine whether Rad10-YFP recruitment is diminished in the absence of SAW1.
Cloning was carried out by construction of DNA plasmids, each containing portions of the sequence needed to install the appropriate DSB substrates. Plasmids were then linearized and transformed into the yeast genome. The resulting strains contain an I-SceI restriction enzyme cut site centered between two copies of the HIS3 gene (the DNA repeats) separated by either 20 or 40 bp of nonhomologous sequence on each side (yielding 30 or 50 deoxynucleotide flaps during SSA). Sequencing data confirm that the desired 30 and 50 deoxynucleotide flap SSA substrates have been successfully integrated into the yeast genome. Preliminary microscopy experiments verify the presence of expected fluorescence signals signifying the successful cloning of strains. These strains will allow fluorescence microscopy investigation of Saw1 and Rad1-Rad10 recruitment to SSA sites containing varying flap lengths.
The authors thank NIH grant SC3GM093858 for funding.
Poster #: 184
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: Medicinal Chemistry, Protease inhibitor, Structure activity relationship study
Project Title: Structure activity relationship study of isoleucine sulfonamide hydroxamic acid inhibitors for the Botulinum Neurotoxin
Author List:
Rodriguez Beltran, Sandra; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton, Presenting Author, Nagel Award Finalist
Lien, Thanh; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton
Salzameda, Nicholas; Chemistry & Biochemistry, California State University, Fullerton
Abstract: Botulism, a severe paralytic disease is caused by the botulinum neurotoxin (BoNT) that is produced by the bacterium Clostridium botulinum. The BoNT is composed of heavy (HC) and light (LC) chains. The HC binds to nerve cells and inserts the LC into the cytosol. The BoNT LC, a zinc metalloprotease, cleaves SNARE proteins that disrupt neurotransmission leading to muscle paralysis that can result in death. The neurotoxin is the most poisonous toxin known to man and due to the ease of obtaining BoNT, there are concerns the neurotoxin could be used for bioterrorism. The lack of viable treatment options for large populations requires new therapeutics countermeasures for botulism. Thus, our laboratory is working on inhibiting the BoNT LC as a novel treatment option.
In our previous studies, we designed molecules that were good inhibitors for the BoNT LC. These molecules contained a biphenyl ring attached to the N-terminus of isoleucine via a sulfonamide bond and the C-terminus was functionalized as a hydroxamic acid. We continue our inhibitor study for the BoNT LC focusing on the biphenyl and the importance of the sulfonamide. The sulfonamide linker is key to many therapeutics and we are interested to learn if the sulfonamide is also vital for BoNT LC inhibition. In addition, we sought to study how the number and position of chlorines on the biphenyl affects inhibition. These compounds were synthesized through a 4 step synthetic route beginning with isoleucine included an sulfonamide or amide coupling, a Suzuki couplings to build the biphenyl structure and hydrolysis of the methyl ester to a hydroxamic acid.
Based on an enzymatic BoNT LC inhibition assay, the position of the chlorine was important for inhibition. Inhibitors that contained at least 1 chlorine at the meta position of the biphenyl displayed better inhibition then other analogs, with the best compound having an IC50 value of 20 µM for the BoNT LC. Interestingly when the biphenyl was replaced with a furan-phenyl structure the inhibition increased with an IC50 of 12 µM. When comparing the inhibition of compounds containing the sulfonamide or amide linker it was determined that the amide linker displayed better inhibition for the BoNT LC. The best inhibitor containing the amide linker and biphenyl with chlorine at the meta position had an IC50 of 5 µM for the BoNT LC. Based on the results we have a better understanding of small molecule structural requirements for BoNT/LC inhibition.
Poster #: 185
Campus: San José State University
Poster Category: Bioengineering
Keywords: Chlamydia pneumoniae, Atherosclerosis, Biophysical forces
Project Title: Biophysical forces modulate Chlamydia pneumoniae-exacerbated atherosclerosis
Author List:
Ramasubramanian, Anand; Biomedical, Chemical, and Materials Engineering, San José State University, Presenting Author
Evani, Shankar; University of Texas at San Antonio
Dallo, Shatha; University of Texas at San Antonio
Abstract: Atherosclerosis is a chronic inflammatory disease. In addition to well-documented genetic and environmental factors, there is compelling epidemiological evidence that, either directly or indirectly, microbial infections (‘infectious burden’) play an important role in the development and progression of atherosclerosis. Specifically, multiple lines of evidence implicate that Chlamydia pneumoniae infection is a highly likely risk factor for atherosclerosis, based on several in vitro, seroepidemiological, histopathological, animal models, and limited clinical intervention studies. While the pathogenic role of C. pneumoniae as risk factor in atherosclerosis is widely accepted, the exact mechanistic contribution is poorly understood, thus limiting options for anti-infective prevention or intervention. Of interest, local blood flow dynamics and vessel wall compliance are important determinants of atherosclerosis suggesting that, in addition to chemical stimuli, mechanical cues play critical role in disease progression. We hypothesized that the biochemical and biomechanical effects of C. pneumoniae infection in the vasculature and in the vascular wall microenvironment hasten atherosclerosis. Using an in vitro model of blood flow and vascular wall to live and heat-killed C. pneumoniae infection, we show that: (i) exposure of infected macrophages with to physiological levels of shear stress showed significant increase in inflammatory cytokines (TNFα, IL-1β and IL-8) production compared to infected cells alone; (ii) sheared and infected macrophages show increased expression of endothelial adhesion receptors, and increased adherence to endothelial cells under flow, compared to infected cells alone; (iii) infected macrophages remodel collagen matrix as evidenced by changes in fiber density, viscous and elastic moduli, and release of matrix-degrading proteases and reactive oxygen species; and (iv) infected macrophages lodged in the matrix recruit fresh monocytes, promote efferocytosis, and readily take up LDL to form foam cells. In summary, the results will provide crucial information on C. pneumoniae as a risk factor in plaque formation, and also serve a general framework for evaluating the interplay between chemical and mechanical stimuli in microbial vascular infections and associated inflammation. Acknowledgement: This work is funded by NIH grant HL112629
Poster #: 186
Campus: CSU Northridge
Poster Category: Bioengineering
Keywords: Aneurysm Blood Vessel, 3D bioprinting, Cardiovascular Fluid Dynamics
Project Title: 3D Bioprinting of Aneurysm Blood Vessel and its Fluid Dynamic Analysis
Author List:
Aguiar, Daniel; Graduate, Manufacturing Systems Engineering & Management, California State University, Northridge, Presenting Author
Bermudez, Juliana; Undergraduate, Manufacturing Systems Engineering & Management, California State University, Northridge, Presenting Author
Yu, Paulo; Graduate, Mechanical Engineering, California State University, Northridge
Durgesh, Vibhav; Mechanical Engieering, California State University, Northridge
Li, Bingbing; Manufacturing Systems Engineering & Management, California State University, Northridge
Abstract: 3D bioprinting has opened the door to new methods of printing, especially the methods used for tissue engineering and the production of artificial organs and tissues. Having the capability to 3D print functional artificial tissues on demand is extremely beneficial for studying all aspects of these tissues and organs and ultimately eliminating the need for donors. Cardiovascular disease is the leading cause of death in the United States, at a staggering 25% of fatalities for both men and women. Aneurysms alone caused more than 9,000 deaths in 2014 and two-thirds being male.
A better understanding of the flow parameters that causes rupture is crucial. For this reason, the production of an aneurysm blood vessel model can allow researchers to find new methods of testing and analyzing this naturally occurring abnormality. We demonstrated the possibility of printing a healthy blood vessel as well with vascular aneurysm scaffolds created by the FRESH Method (Freeform Reversible Embedding of Suspended Hydrogels). By using a thermoreversible crosslinking supporting material composed of gelatin and CaCl2, that works as a support material for the alginate hydrogel. The hydrogel bioink is deposited by an adapted XYZ stage and a syringe extrusion dispenser. The support material is then dissolved in a warm bath of a higher concentration of CaCl2 and H2O where the hydrogel crosslinking process is then finalized. Once the gelatin support is fully dissolved, the printed vessel model is fully crosslinked and ready to be removed from the bath. The fluid dynamics parameters in these models was studied by using Laser Doppler Velocimetry and Particle Image Velocimetry measurements.
Funding agency support:
NIH NIGMS BUILD (Building Infrastructure Leading to Diversity) PODER (Promoting Opportunities for Diversity in Education and Research) Faculty Scholar Academy and Mentor Training (1RL5GM118975), Equipment (8UL1GM118976) and Student (BTL4GM118977).
NSF CSU I-Corps Site (California State University Innovation Corps) Faculty Lead Microgrant (57866AP38147811213).
Poster #: 187
Campus: Humboldt State University
Poster Category: Bioengineering
Keywords: hydrogel, chitosan, scaffold
Project Title: Biorenewable Hydrogel Polymers from modified Chitosan: Progress toward three dimensional cell growth scaffolds
Author List:
Sehawneh, Diana; Undergraduate, Chemistry, Humboldt State University, Presenting Author
Alizadeh, Tara; Undergraduate, biology, Humboldt State University, Presenting Author
Jacobs, Jordan ; Undergraduate, chemistry, Humboldt State University
Ward, Mitchell; Undergraduate, Chemistry, Humboldt State University
Cappuccio, Frank; Chemistry, Humboldt State University
Abstract: Hydrogel polymers have found widespread application in many fields. They are used as absorbents, contact lenses, drug delivery systems, and in biomedical applications such as tissue engineering and dissolvable sutures. Hydrogels are water-containing gels that have a hydrophilic polymer network and can absorb a large quantity of water without the dissolution of the polymer network. The current research focuses on producing hydrogels from chitosan, a bio-renewable polymer. Chitosan is the deacetylated form of chitin, which is a polysaccharide produced from β-1,4-N-acetyl-D-glucosamine. Chitin is the second most abundant biopolymer on earth found in the exoskeletons of crustaceans, such as shrimps, crabs, and the cuticle of insects. Chitosan is non-toxic, biocompatible, and biodegradable.
Amino acid coupling, using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), is employed to attach trans-cinnamic acid to the free amine of the amino sugar backbone. The alkene group present in trans-cinnamic acid provides a cross-linkable unit to the chitosan chain. The resulting solutions are photocrosslinked in a catalyst-free environment under broad band ultra-violet (UV) radiation to produce hydrogels. The hydrogels show an average 75% swelling capacity in H2O, and are being evaluated for sterilization and as scaffolds for three dimensional cell growth studies. Experiments are currently underway to incorporate Laminin derived protein sequence (-GGSDPGYIGSR-) or bone marrow homing factor derived sequence (-GGPFSSTKT-), which have been shown to be supportive of neuronal cell differentiation, or other biomimetics into the hydrogels.
Poster #: 188
Campus: CSU Fullerton
Poster Category: Bioengineering
Keywords: Electrooculography, ALS, EOG
Project Title: EOG Eye Tracker for Computer Control
Author List:
Larson, Alex; Undergraduate, Computer Engineering, California State University, Fullerton, Presenting Author
Herrera, Joshua; Undergraduate, Computer Engineering, California State University, Fullerton, Presenting Author
O’Bard, Bryce; Graduate, Computer Engineering, California State University, Fullerton
Mangukiya, Yamik; Graduate, Computer Engineering, California State University, Fullerton
George, Kiran; Computer Engineering, California State University, Fullerton
Abstract: Computers are without a doubt one of the most important devices that we interact with on a day to day basis. We use them to create, express ourselves, and communicate with the world. Unfortunately, nearly one million individuals in the United States are affected with paralysis, loss of motor skills, motor neuron disease such as ALS. As a result, there are many people who are unable to operate a computer or other electronic devices. This study presents an electrooculography (EOG) based device that allows a user to perform basic computer tasks by recording eye movement. Electrooculography is a biosignal which is found by measuring the corneo-retinal potential using a series of electrodes placed around the eye. The electrodes are connected to a circuit we designed which amplifies and filters before they are fed to an analog-to-digital converter connected to a microcontroller which transfers the data to a PC for further processing. The real challenge of EOG lies in processing this data, this can be difficult due to inconsistent nature of the signals and their tendency to drift over time. To overcome these shortcomings we developed a unique algorithm that is able to distinguish eye movements from the noise and drift that affects the EOG signals. Using this algorithm we are able to reliably determine in which direction the user is looking. In order to operate the computer the user simply needs to look in a direction and the mouse will move at a constant rate in that direction. To click on an object the user simply holds their gaze on the screen for a few seconds. With practice a user can perform basic computer tasks such as opening web pages and typing short messages. The effectiveness of our device was determined through a series of trials on 5 healthy subjects. The test subject were tasked with moving the cursor among a series of 9 on screen shapes and the time taken to move to each location was recorded. To limit the length of the trials a task was considered failed if the user was unable to complete it within a one minute timeframe. The average success rate across the 5 subjects we tested was 90.0% and the average time taken to complete the test was 214 seconds. We ran the test twice for each subject and saw an average improvement of 12.16% in the second test, showing that the effectiveness of the device can increase with practice. These test results suggest that EOG shows promise as low cost method to allow those with disabilities to operate a computer.
Poster #: 189
Campus: San José State University
Poster Category: Bioengineering
Keywords: BRAF-V600E, HIV-Integrase, Machine Learning
Project Title: Comparing The Ability Of 3D-QSAR and SVM Models To Predict BRAF-V600E and HIV Integrase Inhibitors
Author List:
Veerapaneni, Saihitha; Graduate, General Engineering, San José State University, Presenting Author
Desai, Rachana; Graduate, General Engineering, San José State University, Presenting Author
Joglekar, Namrata; Graduate, General Engineering, San José State University
McGee, Francisco; Graduate, General Engineering, San José State University
Rao, Sheela; Advent Engineering Services
Wesley, Leonard; Computer Science, San José State University
Abstract: 3D quantitative structure–activity relationship (3D-QSAR) models and support vector machine (SVM) models are two of many machine learning (ML) technologies that are used to help with drug discovery and development task. By quantifying the relationship between molecular structure and extrinsic biological assay data for a set of known inhibitors, 3D-QSAR and SVM models can be trained to predict biological activity for potential inhibitors which have not yet been assayed. A frequent question when presented with such prediction tasks is which of the two models is better for the challenge at hand. Here we present the results of directly comparing the ability of 3D-QSAR and SVM models to predict the activity of 303 analogs (68 active) of Vemurafenib® (PLX4032), an FDA-approved treatment for melanoma and other cancers, against the BRAF-V600E protein. We also present the results of directly comparing the ability of 3D-QSAR and SVM models to predict the activity of 204 small molecule compounds (159 active) to inhibit HIV Integrase. This work is motivated by the fact that previous 3D-QSAR and SVM comparative work did not use the same targets and compounds for each model, or only the training and cross validation metrics were reported and actual prediction accuracy was not carried out.
3D-QSAR models for BRAF-V600E and HIV Integrase were built using the molecular modeling software Molecular Operating Environment (MOE) marketed by the Chemical Computing Group. SVM models for the same targets were built using scikit’s sklearn ML package. 60 of the 303 Vemurafenib® analogs were set aside for testing, and 41 of the 204 potential HIV Integrase inhibitors were set aside for testing. Models were trained and optimized with respect to R2, Q2, MSE, cross validation measures and so forth. A SVM prediction accuracy of 95% (BRAF-V600E) and 100% (HIV Integrase), and 3D-QSAR prediction accuracy of 76% (BRAF-V600E) and 82% (HIV Integrase) was observed. The results indicate that the SVM classifier performed over 15% better than the 3D-QSAR classifier for both targets. A partial explanation of why the 3D-QSAR model did not perform as well as the SVM classifiers is that it did not have a comparable and theoretic “shatter capacity” measure as that of the SVM models. In conclusion, an optimized SVM classifier can be expected to perform significantly better than an optimized 3D-QSAR model when predicting BRAF-V600E and HIV Integrase inhibitors.
Poster #: 190
Campus: CSU Fresno
Poster Category: Bioengineering
Keywords: lead, bone, x-ray fluorescence
Project Title: Measurement of soft tissue thickness using Lβ/Lα ratio in L-shell x-ray fluorescence measurements of lead in bone and soft tissue phantoms
Author List:
Gherase, Mihai; Physics, California State University, Fresno, Presenting Author
Freire-Gama, Andressa; Undergraduate, Physics, California State University, Fresno
Abstract: Lead (Pb) is a well-known toxic element. Its adverse effects following prolonged exposures are known for a long time. However, recent research correlated detrimental effects on cognitive abilities in children with very low blood level concentrations (
Poster #: 191
Campus: CSU Fullerton
Poster Category: Bioengineering
Keywords: mirror neuron, virtual reality, upper limb rehabilitation
Project Title: Development of a Virtual Reality Environment for Upper Limb Rehabilitation for Post-Stroke Patients
Author List:
Ahir, Vishalkumar; Graduate, Mechanical Enginerring, California State University, Fullerton, Presenting Author
Faller, K. J.; Computer Engineering, California State University, Fullerton, Presenting Author
Garrett, N.; California State University, Fullerton
Robson, Nina; Mechanical Engineering, California State University, Fullerton
Abstract: Retraining the motor system of subjects suffering partial loss of motor ability due to strokes is a task that presents a number of difficulties. In many cases, only partial success is accomplished after long training sessions. The discovery of mirror neurons over a decade ago has led to extensive research that has revealed a neural network that supports humans’ ability to learn through action imitation and observation. Currently the training of individuals that has suffered from stroke is done using mirrors. While the mirror therapy is suggested to be a simple, inexpensive and patient-directed treatment that may improve upper-extremity function for sub-acute stroke patients, it has been shown that it does not affect patients with spasticity, as well as does not offer an accurate and consistent assessment of the patients’ condition during and post-therapy.
The aim of this research was the development of an augmented reality wearable device that creates a virtual reality environment for the human arm in order to increase the success of training and relearning. The device performs a symmetric reflection and real time mapping of the stroke patients’ healthy limb onto the most affected limb. Using the device, our experiments tested the impact of the existing mirror therapy compared to the developed virtual reality environment on four healthy subjects, between the ages of 20 and 50. During testing the subjects were required to use their non-dominant upper extremity to employ static and dynamic tasks for a total of 10 trials using both mirror and virtual reality therapy. At the end, the subjects were required to fill out an anonymous survey regarding their experiences. The tests and survey showed that the virtual reality training provides a more diverse and engaging environment. In addition, the virtual reality training allowed for and bi-manual task setup, as well as for the extraction of precise experimental data related to the upper extremity timing, trajectory, velocity, etc. that were used in the post-training analysis.
Based on these promising results, using the developed virtual reality system, the next steps involve the development, testing and assessment of effective action-observation relearning protocols that may benefit the recovery of function after a stroke, enhance clinical training protocols, and lead to transfer of recovered function from clinic to Activities of Daily Living.
Funding: General and Age Related Disabilities Program, NSF
Poster #: 192
Campus: CSU Northridge
Poster Category: Bioengineering
Keywords:
Project Title: Computational Investigation of Nano Fluidic Porous Filled Heat exchangers for Thermal
Author List:
Zing, Carlos; Graduate, Mechanical Engineering Department, California State University, Northridge, Presenting Author
Mahjoob, Shadi; Mechanical Engineering Department, California State University, Northridge
Abstract: Thermal management has an important role in the development of advanced biomedical devices, due to high usage of electronics and micro processors in these devices. In order to keep the temperature below a maximum operating temperature, cooling techniques and temperature control have become an essential need for variety of high performance medical devices such as imaging equipments, PCR (polymerase chain reaction) machines, lasers, ultrasound equipments, radiography machines and surgical instruments. In this work, a porous filled heat exchanger has been simulated numerically to investigate the effects of usage of different water based nano-fluids (5% double walled carbon nanotube in water (DWCNT), 1% alumina in water, and 1% diamond in 20:80 ethylene glycol/water) as coolant in heat transport improvement [1-2]. The study has been performed for two different porous solid materials (copper and annealed pyrolytic graphite (APG)), at low and high porosity values. High conductive porous inserts provide a large surface area for a given volume that is an essential parameter in heat transfer augmentation and thermal control [3-6]. Comparing the two investigated materials, APG is a lighter and more conductive material, but fragile in comparison with copper. In order to cool the biomedical/electronic devices, the base of the porous filled heat exchanger will be in contact with those devices and so the base will be subjected to high heat flux leaving the devices. The results indicate that utilizing APG porous matrix, for all studied coolants of pure water and water based nano fluidics, improves substantially the cooling of the base of the heat exchanger and so the biomedical/electronics device. Also APG porous matrix provides a more uniform temperature distribution on the base. Comparing the investigated coolant fluids, diamond nanofluids has lower thermal effectiveness in comparison with pure water and other studied water based nanofluids. However, the results indicate that utilizing carbon nanofluids (DWCNT) as coolant for high porosity structures, in both copper and APG porous matrices, improves cooling efficiency and temperature uniformity over the base and so the biomedical/electronic devices.
Poster #: 193
Campus: CSU Los Angeles
Poster Category: Clinical
Keywords: Antimicrobial Lipids, Innate Immunity, Histochemistry
Project Title: Assessing lipid accumulation in airway mucosa from patients with chronic rhinosinusitis
Author List:
Pollock, Nicolette Pollock; Undergraduate, Biological Sciences, California State University, Los Angeles, Presenting Author, Nagel Award Finalist
Alvarez, Sandy; Graduate, Biological Sciences, California State University, Los Angeles
Lee, Jivianne; Orange County Sinus Institute, SCPMG, Irvine, California
Porter, Edith; Biological Sciences, California State University, Los Angeles
Abstract: Background: Epithelial cells lining the respiratory tract contribute to the first line of mucosal defense through the production of antimicrobial peptides and initiation of the adaptive immune response. Recent evidence suggests that antimicrobial lipid production is induced in sinus mucosa from patients with chronic rhinosinusitis (CRS) but the cellular source of these lipids is unclear. Macrophages have been shown to accumulate in inflamed and infected tissues, further enhancing the inflammatory immune response and are also known to form lipid rich foam cells in atherosclerosis. Objective: To assess lipid accumulation in formalin fixed sinus tissues from patients with and without chronic rhinosinusitis with the lipophilic red fluorescent dye Nile Red. Results: We found that in most of the non-CRS controls, the respiratory epithelium was more prominently stained with Nile Red whereas in most of the CRS samples, submucosal tissue was more prominently stained. This was mirrored by ImageJ relative fluorescence quantification (means ± SEM for epithelium 266.35 ± 68.21 vs 213.46 ± 32.17, and for submucosa 493.51 ± 41.25 vs 623.55 ± 89.10, for non-CRS (n = 7) vs CRS (n = 17), respectively. However, these differences were not statistically significant, most likely due to clinical subgroups of CRS requiring more tissue samples to be analyzed. In addition, the submucosa of CRS subjects contained numerous cells with granular lipid accumulation, reminiscent of macrophages. Co-localization studies employing macrophage specific probes have begun. Conclusion: In CRS, macrophages may be recruited to the site of infection delivering antimicrobial lipids. Acknowledgements: NIH T34 GM00822 and NIH 1SC1GM096916
Poster #: 194
Campus: CSU Long Beach
Poster Category: Clinical
Keywords: Addiction, Ritalin, Animal models
Project Title: Methylphenidate Exposure Early in Development Alters Oxycodone Reward in Male and Female Adolescent Rats
Author List:
Sollenberger, Nathan; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Manoogian, Adam; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Park, Hyejin; Undergraduate, Psychology, California State University, Long Beach
Zavala, Arturo; Psychology, California State University, Long Beach
Abstract: Methylphenidate (MPH) is commonly prescribed to treat the symptoms for ADHD in preschool-age children. However, its long-term side effects remain unclear. Preclinical research has shown that MPH pretreatment during postnatal days (PDs) 11-20, a period of rat development comparable to preschool-age children, enhances the rewarding effects of morphine when rats are tested as adults. However, the effect of MPH on oxycodone (OXY), a commonly prescribed drug for the management of pain, has not been previously examined. OXY is of particular interest because of the increase in abuse rates among adolescents in recent years. We hypothesized that the rewarding effects of oxycodone, as measured by the validated conditioned place preference (CPP) paradigm, would be enhanced in male and female rats pretreated with MPH early in development. Male and female rats were intraperitoneally injected with either saline or 4 mg/kg MPH twice-daily from PDs 11-20. Rats were then assessed for OXY CPP using an 11-day procedure beginning on PD 40. During a preconditioning session (PD40), rats were tested for their baseline place preference within a two-chamber apparatus in a 15-min session. During conditioning (PD 42-47), rats underwent daily 30-min sessions, which involved rats receiving daily alternating oxycodone (0, .033, 0.1, 0.3 mg/kg) or saline injections in each of the two chambers, respectively. A final postconditioning preference test took place during a 15-min session one day after the end of the conditioning phase (i.e., PD 48). An increase in preference for the OXY-paired compartment was evidence of OXY-induced CPP. MPH pretreatment altered OXY-induced CPP, with MPH producing more robust CPP in a dose-dependent manner. These findings are in agreement with previous data demonstrating an effect of MPH on morphine CPP. Collectively, the use of MPH in preschool age children may enhance susceptibility for opioid drugs later in development, and thus the prescription of MPH should be considered a last resort when working with preschool-age children.
Poster #: 195
Campus: Humboldt State University
Poster Category: Clinical
Keywords: Agining, Walking economy, Bicycling
Project Title: Bicycling for Exercise Helps Retain a Youthful Walking Economy in Older Adults
Author List:
Aslan, Daniel; Graduate, Kinesiology, Humboldt State University, Presenting Author
Collette, Joshua; Undergraduate, Biology, Humboldt State University
Ortega, Justus; Kinesiology, Humboldt State University
Abstract: Introduction: Impaired walking performance is a key determinant of morbidity among older adults [1]. A distinctive characteristic of impaired walking performance among older adults is a greater metabolic cost (worse economy) compared to young adults [2,3]. Specifically, healthy older adults have been shown to have a 15-20% greater metabolic cost of walking compared to young adults. However, a recent study suggests that older adults who routinely run for exercise have an improved walking economy compared to older adults who walk. Perhaps even more impressively, older runners have a walking economy similar to healthy young adults [4]. Yet, it remains unclear if other aerobic exercises such as bicycling elicits similar improvements in walking economy among older adults. Purpose: To determine if regular bicycling exercise affects walking economy in older adults. To our knowledge, there has been no research looking at metabolic cost of walking in older adults who cycle for exercise. Methods: 17 young adults (≈24 years), 17 “older walkers” (≈71.54 years) who walk ≥30 min, 3x/week, and 17 “older cyclist” (≈68.4 years) who cycle ≥30 min, 3x/week, walked on a level treadmill at four speeds (0.75, 1.25, 1.60, 1.75 m/s). Using an open circuit expired gas analysis, we measured metabolic cost (joules/kilogram/second) in the last 2 minutes of each 6 minute trial to determine walking economy. We compared walking economy in the three groups. Main findings: Older cyclists had a 5-12% better walking economy than older walkers and similar walking economy to young adults across all speeds. Conclusion: Bicycling exercise mitigates the age-related deterioration of walking economy, whereas walking for exercise appears to have a minimal effect on the walking economy of older adults. We suspect the greater aerobic intensity of bicycling exercise may maintain muscle mitochondrial efficiency in aging and thus explain the better walking economy in older cyclist versus older walkers.
1. Studenski S, et al. JAMA 305, 50-58, 2011.
2. Martin PE, et al. Journal of Applied Physiology 73, 200- 206, 1992.
3. Ortega JD & Farley CT. Journal of Applied Physiology 102, 2266-2273, 2007.
4. Ortega, JD, et al. PLOSOne, 9(11), e113471. doi:10.1371/journal.pone.0113471
Poster #: 196
Campus: CSU Northridge
Poster Category: Clinical
Keywords: cancer, target, stemness
Project Title: ITGA1 is a novel biomarker and therapeutic target for blocking stemness in pancreatic cancer
Author List:
Kim, Sa La; Undergraduate, Biology, California State University, Northridge, Presenting Author, Nagel Award Finalist
Gharibi, Armen; Graduate, Biology, California State University, Northridge
Brambilla, Daniel; Undergraduate, Biology, California State University, Northridge
Adamian, Yvess; Graduate, Biology, California State University, Northridge
Hoover, Malachia ; Graduate, Biology, California State University, Northridge, Presenting Author
Lin, Joy; Washington University in St. Louis
Agajanian, Megan; Graduate, Biology, California State University, Northridge
Wolfenden, Laurelin; Undergraduate, Biology, California State University, Northridge
Kelber, Jonathan ; Biology, California State University, Northridge
Abstract: Pancreatic ductal adenocarcinoma (PDAC), also known as pancreatic cancer, is the fourth leading cause of cancer-related deaths in the United States with a five-year survival rate of approximately 7%. Its highly invasive characteristics, the lack of reliable biomarkers for early detection, limited therapeutic targets and inherent resistance to available therapeutic interventions (ie., erlotinib, gemcitabine) are the central factors that lead to an overall poor prognosis for this malignancy. Since pancreatic cancer-related deaths are primarily a result of metastatic tumors, we reasoned that our previously published proteomic signature of the cell pseudopodium (PD) may contain potential molecular targets for blocking pancreatic cancer. From a series of bioinformatic analyses, we identified ITGA1 as a candidate for further testing. We first established that ITGA1 is upregulated in PDAC using cell line models as well as banked microarray and IHC data. Importantly, we have also discovered pre-malignant upregulation of ITGA1 in pancreatic intraepithelial neoplastic lesions (or PanINs) suggesting its utility as an early indicator of cancer initiation in the pancreas. To evaluate the function of ITGA1 in PDAC, we generated knockdown lines in two PDAC cell models. Using these modified cells, we have demonstrated that ITGA1 is necessary for pancreatic cancer cell survival, migration and adhesion/spreading on collagen substrates. Tumor-initiating cells (or stem-like cancer cells) have been identified and characterized in pancreatic cancer to be positive for aldehyde dehydrogenase activity and to contribute therapeutic resistance. However, the role of ITGA1 in these tumorigenic cell populations has not yet been characterized. Our results further indicate that ITGA1 is required to maintain cell viability in the ALDH1hi cell population of PDAC cells. Taken together, our data suggest that ITGA1 is a viable indicator of tumor initiation and therapy resistance, as well as a novel molecular target in pancreatic cancer.
Poster #: 197
Campus: Humboldt State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Food competition, Acoustic signaling, Bats
Project Title: Sing or Jam? Density-dependent food competition strategies in Brazilian free-tailed bats (Tadarida brasiliensis)
Author List:
Tamura, Shin; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
Gotgotao, Jeremy; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
Corcoran, Aaron; Biological Sciences, Humboldt State University
Abstract: Limited food resources and competition have driven animals to evolve food defense mechanisms. The Brazilian free-tailed bat (Tadarida brasiliensis) is a useful model species for studying food competition because it lives in the largest colonies of any mammal. Previous research has demonstrated that T. brasiliensis uses specialized acoustic signals (sinFM calls) to jam the echolocation of conspecifics attempting to capture insects. Male T. brasiliensis also make individual-specific songs, which are used in roosts to attract mates and fend off other males. Preliminary observations indicate that T. brasiliensis also produce song at feeding sites, but the function of song in this context is unknown.
We used infrared videography and ultrasonic audio recordings to document T. brasiliensis foraging and social behavior at two field sites in Arizona and New Mexico. Calibrated, multi-camera video recordings allowed 3-D reconstruction of bat social interactions. Continuous ultrasonic audio recordings made over 10 nights allowed us to correlate patterns of bat activity (through echolocation call counts), feeding activity (number of feeding calls made), song production and sinFM call production. Insect activity was monitored simultaneously with acoustic recordings using ultraviolet light traps.
Linear mixed effects statistical models indicated that bats sing more often when there are relatively few competing bats present, and jam more often when there are high numbers of competing bats present. Insect abundance was not correlated with bat activity levels, song production or sinFM production. 3-D videography revealed bats frequently chase conspecifics while singing. No chases were observed for bats jamming conspecifics.
Our results suggest that bats sing as a mechanism for territorial food defense when competition levels are low, and switch to jamming competing bats when competition levels increase above a set threshold. This indicates that bats may be unable to maintain food territories in the presence of multiple competitors, and have had to evolve another strategy (jamming) for this situation. The density dependent use of either song or jamming demonstrates the behavioral versatility of Brazilian free-tailed bats, and the high level of food competition present in this species. These results may be of interest to roboticists interested in the interactions of competing robots.
Poster #: 198
Campus: CSU San Marcos
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Genomics, Population genetics, Bioinformatics
Project Title: PPP v.1.0 – Population Genomics Pipeline Platform – A Galaxy based pipeline for model-based population genomics
Author List:
Sethuraman, Arun; Biological Sciences, California State University San Marcos
Takehara, Shiori; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Lynch, Melissa; Undergraduate, Biological Sciences, California State University San Marcos
Hey, Jody; Temple University
Abstract: The field of population genomics, or using large scale genomic data to inform diversity and variation in natural populations has grown tremendously in the last decade. This is owed in part to advances in genomic sequencing technologies which allow researchers access to large amounts of data at consistently plummeting costs, and in part to the development of sophisticated statistical methods to analyze this data. However, there is yet to be a comprehensive, easy to use software pipeline that fills this gap between data and results in population genomics. The PPP (Population Genomics Pipeline Platform) is a multi-year collaborative project funded by the NSF being developed by researchers at the CSU San Marcos and Temple University (Philadelphia). Here we present the first phase of the PPP, which implements scripts for analyzing genomic variation, pruning data, and running model-based isolation with migration (IM) analyses on the Galaxy Project. Future builds of the PPP will implement advancements including data clean-up, building evolutionary models, bioinformatics analyses and data visualization. Latest builds of the PPP can be accessed via http://www-dev3.cst.temple.edu/.
Poster #: 199
Campus: Sacramento State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: electrochemistry, density functional theory (DFT), organometallic
Project Title: Mechanism for the Stereoselectivity in Metal-Salen Catalyzed Electroreductive Cyclization Reactions
Author List:
Bellini, Leonard; Graduate, Chemistry, California State University, Sacramento, Presenting Author
Cihak, Michelle; Undergraduate, Chemistry, California State University, Sacramento
Silva, Braden; Undergraduate, Chemistry, California State University, Sacramento
Gherman, Benjamin; Chemistry, California State University, Sacramento
Miranda, James; Chemistry, California State University, Sacramento
Abstract: The total synthesis of natural products with pharmaceutical applications, such as the Corey lactone derivative (a prostaglandin precursor) and quadrone (an anticancer compound), are facilitated by reactions that couple functional groups of similar polarities. This type of reaction can be realized by the use of electroreductive cyclization (ERC). ERC reactions occur, for example, when the electron-deficient β carbon of an α,β-unsaturated ester is reduced, allowing it to react with an electrophilic site such as the carbonyl carbon of an aldehyde or ketone or the β carbon of a second α,β-unsaturated ester. If both of these functional groups are part of the same molecule, the new carbon-carbon bond will result in cyclization. Electron transfer to the ERC substrate can be mediated by the use of metal-salens. Previous theoretical work showed that Ni(II)-salen and Zn(II)-salen favor inner-sphere electron transfer. This computational study focuses on the mechanism and stereoselectivity of substrate cyclization in ERC reactions following the electron transfer to substrate from both chiral and achiral Ni(II)-salen and Zn(II)-salen. Two reaction pathways are examined for the cyclization of the ERC substrates, an α,β-unsaturated ester and aldehyde. One is a concerted pathway in which cyclization and separation of the substrate from the metal-salen is simultaneous. This concerted pathway is essential for directing the stereoselectivity of the product during the reaction and substantiates the utility of ERC in organic synthesis. The other is a stepwise pathway in which the substrate first detaches from the metal-salen. The resulting intermediate radical carbanion then proceeds to cyclize, yielding equal amounts of both cis and trans isomers. The computed energetics for the ERC reactions, and the kinetically preferred pathway for each salen/substrate combination is determined using density functional theory (DFT) calculations. In the case of the achiral metal-salens, the data show an energetic preference for the stepwise pathway over the concerted pathway. Consequently, ERC with the achiral salens is predicted not be stereoselective. ERC catalyzed by chiral metal-salens, however, alters the reaction energetics and gives the possibility for stereoselective cyclization of the substrate.
Poster #: 200
Campus: San José State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: statistical potentials, RNA-protein interactions, docking
Project Title: Second-Generation RNA-Protein Statistical Potentials: Additional Refinements by Structure Class and Atom Type
Author List:
Soshnikov, Artem; Graduate, Chemical Engineering, San José State University, Presenting Author
Kimura, Takayuki; Graduate, Chemistry, San José State University, Presenting Author
Rodriguez, Manuel; Graduate, Chemical Engineering, San José State University
Le, Thanh; Graduate, Chemistry, San José State University
Park, Amos; Volunteer at SJSU, Chemistry, San José State University
Yu, David; Undergraduate, Chemistry, San José State University
Lustig, Brooke; Chemistry, San José State University
Abstract: Computational modeling of RNA-protein interactions is an important tool for biomedical research. However, exclusively all-atom methods that model RNA-protein interactions by molecular dynamics and related approaches are typically problematic. One alternative implements a hierarchical methodology, first exhaustively exploring configurational space with a reduced representation of the RNA and protein. Then, the lowest energy cohort of such coarse-grained models can be used as a scaffold for all-atom placements, a method often implemented in modeling protein 3D-structure. However, the coarse-grained modeling likely will require improved ribonucleotide-amino acid potentials when applied to coarse-grained structures. As a first step we downloaded 1345 PDB files and clustered them with PISCES to obtain a non-redundant RNA-protein complex data set. The contacts were divided into nine types with DSSR according to the structure classification of the RNA component of the interaction (e.g. A-form major-groove base) and then nine sets of potentials were calculated. As expected, the strongest interactions involved Arg-G contacts that specify the RNA bases as opposed to the RNA backbones. Notably, the backbone-related potentials show little individual preference for interaction for a specific nucleotide type among the quartet (A, C, G and U).
The potentials were applied to score fifty thousand poses generated by FTDock for twenty-one standard RNA-protein complexes. The results compare favorably to existing RNA-protein potentials for competitors (Perez-Cano, DARS-RNP and QUASI-RNP), including ranking native structures by energy (percentile rank) among the set of docking poses. And even in the comparison with all-atom density function potentials of ITScore-PR, our 9-term method is not significantly at a disadvantage with its 3.855% mean rank percentile, averaged over the 21-test proteins. Moreover our all-atom version exceeds ITScore-PR, successfully evaluating native-like structures generated by FTDock with a 1.114% mean percentile rank. An even more realistic evaluation of such percentile ranking of potential decoys will likely require relaxation of the rigid protein side chains generated by FTDock, including by molecular dynamics or related methods. In addition, future research will endeavor to optimize potentials that have low frequency of interaction, using improved weighting of such contacts.
Poster #: 201
Campus: CSU Fresno
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Mathematical Biology, Simulation, Foraging
Project Title: Simulation of scene perception, navigation and information storage in wood ants
Author List:
Mendoza, Austin; Undergraduate, Biology, California State University, Fresno, Presenting Author
Arevalo, Erik; Graduate, Biology, California State University, Fresno
Lent, David; Biology, California State University, Fresno, Presenting Author
Abstract: A number of experiments have revealed how different visual features are used to guide familiar foraging routes in wood ants. Using these data, we have developed algorithms to extract visual features that ants use for guidance from panoramic scenes. We have studied how visual cues that ants use are extracted, prioritized and stored during navigation. A foraging model was created in MATLAB to simulate navigation in a procedurally generated environment where the visual cues could be precisely characterized. In these environments, our algorithms extracted and stored the visual cues that were available during a single Levy walk foraging event. Following a random foraging event, the success on subsequent foraging bouts using the stored information was examined. When we examined subsequent foraging walks we found the success of the simulated ant in finding the goal location using only a particular cue or a combination of cues depended on two factors – the length of the route and decay rate of information in a memory network. To further explore this we simulated the Levy walk foraging event over 1000, 2000 and 10000 sampling points and implemented linear or exponential decay in the networks storing the information. Our data suggests that the optimal strategy is to sample and store between 1000 and 2000 points along the foraging route with a network subjected to exponential decay. These parameters resulted in a stored representation that allowed the simulated ant to best find the goal on subsequent foraging bouts. The results of these simulations have provided insight into the mechanisms involved in prioritization and perception of visual information, it supports that ants need only processes relevant cues intermittently and they do not continually process visual information. Additionally, it has let us investigate how learning and storage of spatial information can be optimized in simple networks and nervous systems.
Poster #: 202
Campus: San Diego State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Microbiome, Metagenomics, Binning
Project Title: Optimization of microbiome assembly and binning
Author List:
Papudeshi, Bhavya; Graduate, Bioinformatics and Medical Informatics, San Diego State University, Presenting Author
Matthew Haggerty, John ; Graduate, Biology, San Diego State University
Doane, Michael; Graduate, Biology, San Diego State University
Morris, Megan ; Graduate, Biology, San Diego State University
Beattie, Doug; University of New South Wales, Australia, Biotechnology
Walsh, Kevin; Graduate, Biology, San Diego State University
Gueiros, Genivaldo; Graduate, Computational Sciences, San Diego State University
Edwards, Robert; Computer Science, San Diego State University
Dinsdale, Elizabeth ; Biology, San Diego State University
Abstract: Microbiome/host interactions affects host health. Microbiome description and understanding the effect of disruption to its characteristics (e.g. with antibiotics) is important to medical and agricultural industries. Metagenomics is a technique that provides a description of the microbial community structure and functions. Reconstructing genomes from small fragments of sequences generated by next generation sequencing is complicated due to high variation in diversity across microbiomes. However, new tools are rapidly evolving to group phylogenetically related sequences into bins. The quality of the bins extracted is influenced by metagenome assembly. We have therefore developed a pipeline that performs quality control to both the assembly and binning tools of both high and low diversity microbiomes. The workflow begins with assembly of the metagenomes using three different tools: SPAdes, IDBA, and MetaVelvet. The best contig assembly is binned using MetaBat, and GroopM which extracts bins based on genome signatures, such as tetranucleotide frequencies and contig coverage. Last, the bins are assessed for genome completeness and richness using CheckM and Anvi’o. This pipeline was developed and validated using a low diversity and 3 high diversity microbiomes, a total of 105 metagenomes (15.5 Gbp). Low diversity microbiomes were derived from enriched microbiomes associated with benthic organisms. Two high diversity microbiomes were associated with the kelp forest of Point Loma, CA, USA and one from coral atoll of Abrolhos Bank, Brazil that were sequenced on both Ion Torrent PGM and Illumina MiSeq sequencing platforms. Through repetition of the pipeline over variable microbiomes we conclude the optimal assembler to be SPAdes due to higher contig continuity and lower number of contig chimeras. MetaBat was the optimal binning tool due to higher genome completeness and lower species richness per bin. The optimal workflow for the low diversity samples extracted 17 bins with the highest genome completeness of 94 %. For the high diversity samples resulted 44 and 57 bins from kelp forest and coral atolls were extracted. The bins extracted from the microbiomes enable descriptions of novel genomes, the genes carried by specific taxa, and gene sharing across genera. Using this pipeline across other microbiome, samples such as human microbiomes, will improve our understanding the microbial community and enable targeted interventions.
Poster #: 203
Campus: CSU Long Beach
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Phylogenomics, Phylogenetics, Bioioformatics
Project Title: Large scale bacterial phylogenomics for phylogenetic identification
Author List:
Bezayiff, Darrian; Undergraduate, Computer Science , California State University, Long Beach, Presenting Author
Berlemont, Renaud; Biological Sciences, California State University, Long Beach
Abstract: A fundamental principle of bacterial phylogeny is that closely related strains according to 16S rRNA sequences share most of their functional traits. Conversely, distantly related organisms are expected to display extensive variations in the gene content. Thus, knowing the phylogeny of an isolate, one could potentially predict its functional traits, and vice versa. Here we investigated the correlation between 16S rDNA based phylogeny and the distribution of functional genes in sequenced bacterial genomes. More precisely, we created a custom bioinformatic program aimed at identifying the distribution of functional genes in sequenced bacterial genomes. The distribution of 11,713 gene types was used to cluster 4,116 fully sequenced bacterial genomes. The 16S rRNA-based and gene-content (i.e., phylogenomic tree) based clusterings correlated with each other (p-Mantel = 0.001, r = 0.9628).
Then, subsets of functional genes were sampled randomly to create sub-phylogenomic trees, and compared with the two previously described datasets (16S rRNA tree and phylogenomic tree). Both the size of the subset (the number of traits) and the function of the traits affected the correlation between sub-phylogenomic trees and phylogenomic and the phylogenetic tree. Thus, using phylogenetic information, one can predict some of the functions, and conversely knowing the function of bacteria one could predict it phylogeny.
Poster #: 204
Campus: CSU East Bay
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Chemical imaging, Aptamer-based sensor,
Project Title: An aptamer-based sensor for detecting galactose metabolites
Author List:
Luu, Anthony; Undergraduate, Chemistry and Biochemistry, California State University, East Bay, Presenting Author
Fong, Melissa; Undergraduate, Biological Sciences, California State University, East Bay
Santos, Andrew; Undergraduate, Health Sciences, California State University, East Bay
Nguyen, Nguyen; Undergraduate, Health Sciences, California State University, East Bay, Presenting Author
Sigua, Levi; Graduate, Chemistry and Biochemistry, California State University, East Bay
Chan, Tracy; Undergraduate, Chemistry and Biochemistry, California State University, East Bay
Tang, Chenny; Undergraduate, Biological Sciences, California State University, East Bay
Halim, Marlin; Chemistry and Biochemistry, California State University, East Bay
Abstract: Background: Galactosemia is a genetic disorder that results in the attenuated ability to digest galactose, which in turn, leads to the accumulation of galactose metabolites, i.e. α-D-Galactose-1-phosphate (DG1P), Galactonate (Galn), and Dulcitol (Dul) in the patients’ plasma and urine. These molecules are toxic and their concentrations require frequent monitoring; we aim to develop a convenient tool that can be used to detect and quantify them. Our method is based on single-stranded DNA molecules (aptamers) that will change their optical properties (color or light emission) upon binding to the metabolites. Through an iteration of selection and amplification procedure (called SELEX), we will isolate one aptamer each for metabolite for their selective monitoring.
Method: In each SELEX cycle, aptamers that contain a randomized sequence region of 30 bases are hybridized with a biotinylated 13-base complementary strand (CS). The hybrids are immobilized on a streptavidin-agarose column. A series of buffer washes through the column eliminates the aptamers that are loosely bound to the CS. A solution of the target molecule is then added to allow the aptamers that can bind to the target to dissociate from the CS, and elute out of the column. All buffer and target elutions are amplified via polymerase chain reaction and separated by electrophoresis (on 3% agarose gel). The gel is imaged (with ethidium bromide) to qualitatively determine the aptamer-target binding. The desired result is to have bands that are significantly brighter for the target than for the buffer elutions, indicating greater binding towards the metabolites than the background dissociation of aptamers from the CS. The strand-separated amplicon of the target elution is then used as the starting pool for the next round of SELEX. To isolate high binding affinity aptamers, the target solution concentration is slowly lowered to physiologically relevant levels throughout the multiple rounds.
Results and Conclusion: We have completed between 17 – 33 SELEX cycles using 1 – 0.1 mM Dul and DG1P, and 1 – 0.5 mM Galn. Based on the gel images, we may conclude the presence of aptamers that can bind to their respective target molecules in each of the SELEX rounds performed. The PCR amplification yielded 20 – 60 ng/μL of products. Our goal is to eventually isolate aptamers that can bind 0.01 mM of the molecules.
Acknowledgments: This project is supported by the CSUPERB NIG and the CSUEB Faculty Support Grant.
Poster #: 205
Campus: San Diego State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: laser detection, thirdhand smoke, nicotine
Project Title: Sensitive Laser-Based Detection of Nicotine and its Metabolites for Secondhand and Thirdhand Smoke Studies
Author List:
Brown, Mya; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Munshi, Zarina; Graduate, Chemistry and Biochemistry, San Diego State University
Tong, William; Chemistry and Biochemistry, San Diego State University
Abstract: Ultrasensitive detection methods for nicotine and its major metabolites are demonstrated for studying firsthand, secondhand and thirdhand smoke effects on children. We demonstrated laser wave-mixing spectroscopic methods as sensitive, portable and compact detectors suitable for field use. Reliable detection of nicotine is essential to track and understand numerous health and psychological effects caused by firsthand, secondhand and possible thirdhand smoking. Our results indicated that nicotine and cotinine can be detected and separated in their native forms label-free using a 266 nm UV laser, or using a visible laser if a label is used. In our laser wave-mixing setup, the input laser beam is split into two input beams and then focused and mixed inside the sample cell that contains nicotine or cotinine. Since the signal is a coherent laser-like beam, it can be collected by a simple photodetector with excellent signal-to-noise ratio. Wave mixing only requires a small amount of sample (nanogram), and hence, it can be conveniently interfaced to microarrays, microfluidics, chip-based capillary electrophoresis and other multi-channel flow systems to yield excellent chemical specificity and detection sensitivity levels (picomolar or femtomole). We obtained detection limits better than sub-μg/mL for nicotine and cotinine complexes using a simple 633 nm helium-neon laser. Our detection limits are better than those of currently available methods and our detectors can be used for real-time on-site analyses to accurately monitor smoke exposure to children. Measurement of cotinine in biological fluids would assist in understanding smoke-related diseases.
Poster #: 206
Campus: Cal Poly San Luis Obispo
Poster Category: Diagnostics/Imaging/Analytical
Keywords: skin tissue, γ-H2AX expression, immunofluorescence
Project Title: The Effects of Sphingomyelin on DNA damage due to UV Radiation in Human Keratinocytes
Author List:
Banuelos, Leo; Graduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Graham, Gabriella; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Laiho, Lily; Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Abstract: Non-melanoma skin cancer is the most common type of skin cancer with an estimated 3 million diagnoses every year in the United States as of 2015. Recommended preventative measures include regularly applying sunscreens, but studies into UV filter toxicity in humans are controversial and inconsistent. Furthermore, sunscreens do negatively affect coral reef ecosystems. Alternatives to sunscreens are being studies with sphingomyelin (SM) being seen as a potential molecule for preventing UV damage because of its effects in preventing colon cancer and improving skin barrier function. In this study, to assess SM as possible protection against UV damage in human keratinocytes (KRTs), changes in γ-H2AX expression following 20 mJ of UV exposure were characterized in KRT monolayers grown in an SM-enriched media. γ-H2AX is a phosphorylated histone present at the site of DNA double stranded breaks, therefore it can be used as a measure of DNA damage. The results show a 35% reduction in γ-H2AX expression in SM-enriched KRTs after UV exposure compared to irradiated cells grown in untreated growth media (p < 0.05). This suggests SM offers protection to DNA damage due to UV radiation or allows for faster DNA repair following damage. Future research should characterize γ-H2AX expression following various UV dosages as well as look at changes in ceramide production in cells since ceramide is produce in SM hydrolysis and can act as a cellular messenger for apoptosis.
Poster #: 207
Campus: San José State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: synchrotron radiation, nitrogen vacancy center, cancer detection
Project Title: Using Synchrotron Radiation As a Tool for Bionanotechnology
Author List:
Tran, Polo; Undergraduate, Chemistry, San José State University, Presenting Author
Favre, Elena; Undergraduate, Chemistry, San José State University, Presenting Author
Hernandez, Alejandro; Undergraduate, Chemistry, San José State University
Norlund, Dennis ; Stanford Synchrotron Radiation Laboratory
Wolcott, Abraham; Chemistry, San José State University
Abstract: Here we probe the surface of diamond using synchrotron surface science techniques and exploit this knowledge to engineer a functional nanoprobe. In the field of oncology, there is a need for enhanced cancer detection methods that can verify cancer at the single-cell level. High-pressure high-temperature fluorescent (HPHT) nanodiamonds with nitrogen vacancy centers (NVCs) are an emerging nanoprobe that can detect both electric and magnetic fields. NDs are attractive for biolabeling due to their long electron spin properties, all-carbon matrix, and long-term photostability, but have not been fully utilized as an active sensor in biological systems. The surface chemistry of HPHT NDs plays a vital role in colloidal stability and bioconjugation. This project focuses on the modification of HPHT ND surfaces with amine chemistry through gas phase chemistry. Amination chemistry was carried out in a high temperature tube furnace with reactive gases. Aminated FNDs were characterized with synchrotron-based near edge X-ray absorption fine structure (NEXAFS) and wavelength dependent X-ray photoemission spectroscopy (XPS) and laboratory Raman spectroscopy and FTIR. NEXAFS nitrogen K-edge spectra are suggestive of multiple C-N bond orders. There is evidence of amines (C-N), imines (C=N) and nitriles (CN). Our goal is to form a homogenous amine (-NH2) terminated surface. Amines are a versatile moiety and will provide many routes towards further modification of the ND surface for biolableing. Bioconjugation strategies and cellular staining will then proceed after further verification is established. A key goal is conformation of single antigen-antibody recognition on cancer cell surfaces with NDs using confocal scanning microscopy.
Poster #: 208
Campus: Cal Poly San Luis Obispo
Poster Category: Diagnostics/Imaging/Analytical
Keywords: MicroPAD, Point-of-care Diagnostic, Microfluidics
Project Title: Paper-based point of care diagnostic device design for the quantitative analysis of biological analytes
Author List:
Lore, Brittany A.; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Strong, E. Brandon; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Thiel, Robert; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Martinez, Andres W.; Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo
Martinez, Nathaniel W.; Biological Sciences, California Polytechnic State University, San Luis Obispo
Abstract: The primary objective of this project is to develop a cost-effective, home-use, diagnostic device capable of quantifying biological analytes at femtomolar concentrations. Paper-based diagnostic devices are emerging as inexpensive, reliable, and portable assays capable of providing healthcare professionals with real-time data to ensure accurate diagnosis and treatment. Here we present a first in its kind, quantitative microPAD (μpad) diagnostic test that relies on the deposition of a biological matrix barrier, followed by chronometric detection and quantification of a biological analyte.
Methods: The device design is printed onto Whatman No. 1 CHR paper by wax printing. This process creates hydrophobic barriers, thus resulting in a network of channels and test zones in a 3-dimensional format. Deposition of biological biomatrices, made from mixtures of gelatin and chitosan, result in resistance to solvent passage. Employing a series of on-device competition assays leads to the activation and amplification of pro-enzymes and the ensuing degradation of the biomatrix. Timing the passage of solvent through the 3-dimensional device allows for a corresponding quantitation of analyte. The entire test can be performed in approximately one-hour.
Results: The optimal concentrations of biomatrix range between 4-12% gelatin and 1-2% chitosan. Complete degradation of the biomatrix can be achieved between 5-60 minutes, depending on the initial concentration of analyte. Current ranges of enterokinase (EK) tested, include 14,685pM to 100fM. The devices can be stored at a range of temperatures from -20°C to room temperature, for ~30 days without any observable viability loss.
Conclusion: Deposition of biomatrix on paper based devices allows for the reproducible chronometric quantitation of biological analytes, without relying on enzymatic cholorometric amplification. These biomatrices have proven to be highly stable at wide ranges of temperature and during long periods of time. Given the power of this device, we are currently testing the quantitation of a myriad of serum and salivary biomarkers. Reliable, inexpensive diagnostics will likely prove to be a significant contribution to the diagnostic and healthcare communities.
Funding: CSUPERB New Investigator Award & Presidents’ Commission Scholars Award
Poster #: 209
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: influenza, adjuvant, vaccine
Project Title: Protection Generated Against Influenza in Mice Following Vaccination with Liposomes Containing Only Adjuvants and No Influenza Proteins
Author List:
Villanueva, Paulina; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Guirguis, Fady; Undergraduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Adler-Moore, Jill; Biological Sciences, California State Polytechnic University, Pomona
Abstract: Background: Previous experiments with a liposomal (Lp) influenza vaccine containing both viral protein M2e with the adjuvant Monophosphoryl Lipid A (MPL) or dsRNA, generated a protective adaptive immune response against H1N1 virus. In this study, we examined if mice given liposomes containing only an adjuvant and no protein would stimulate a protective innate immune response against influenza challenge.
Methods: In Study 1,female Swiss Webster (SW) mice (n=10/gp) were vaccinated d0 subcutaneously and intranasally (IN) d28 and d56. Groups were as follows: Lp-MPL-M2e, Lp-Pam3CAG, Lp-dsRNA, Lp-MPL, Lp-1V270, or Lp-lipid Tucaresol. d63. Mice were challenged IN with 10X LD50 H1N1. In Study 2, dsRNA and Pam3CAG were used in a cross protection study against H1N1 and H3N2 challenge. In preparation for Study 2, an LD50 assay was done to determine the H3N2 dilution for the mouse challenge. In Study 2, SW mice (n=12/gp/virus) were vaccinated as in Study 1 with Lp-MPL-M2e-HD, Lp-Pam3CAG, Lp-dsRNA or buffer. d63, mice were challenged IN with a severe H1N1 or H3N2 infection. d69, lungs were collected from 5 mice/gp to determine viral burden using an infectious foci assay. 10 mice/gp in Study 1 and 7 mice/gp in Study 2 were monitored 2X/day for morbidity for 28 days.
Results: In Study 1 survival following H1N1 challenge was 75%, 70% and 63% for Lp-MPL-M2e, Lp-Pam3CAG, Lp-dsRNA, respectively, which was significantly better than the control(10% survival, p<0.016) and better than the liposomes with the other adjuvants. In Study 2, survival against a more severe H1N1 challenge was 57% for Lp-Pam3CAG and 43% for Lp-MPL-M2e-HD versus 0% for the control (p<0.016). With H3N2 challenge, survival with Lp-Pam3CAG(43%) and Lp-dsRNA(29%) was not significantly different than the control(14%)while the Lp-MPL-M2e-HD had 100% survival(p Conclusion: Lp-Pam3CAG and Lp-dsRNA without M2e were as protective against an 10X LD50 H1N1 challenge as Lp-MPL-M2e, but they were not protective against an H3N2 challenge. These results indicate that the liposomes without M2e were stimulating a potent protective innate immune response against H1N1 that was comparable to the adaptive immune response generated by the Lp-MPL-M2e vaccine.
Poster #: 210
Campus: San Diego State University
Poster Category: Disease (Pathogens)
Keywords: EMAST, colorectal cancer, mouse model
Project Title: Discovering a mouse model of EMAST, a biomarker of aggressive colorectal cancer in humans
Author List:
Luu, Jennifer; Undergraduate, Biology, San Diego State University, Presenting Author
Bhaskaran, Nitya; Graduate, Biology, San Diego State University
Mamindla, Priyadarshini; Graduate, Biology, San Diego State University, Presenting Author
McGuire, Kathleen; Biology, San Diego State University
Abstract: Colorectal cancer (CRC) is the one of the leading causes of cancer-related deaths in the US. Elevated Microsatellite Alterations at Selected Tetranucleotide Repeats (EMAST) is a biomarker for aggressive CRC because it is associated with inflammatory responses that promote tumor growth and invasion. EMAST is characterized by insertions or deletions of tetranucleotide repeats in non-coding DNA. Most human studies define a CRC as EMAST positive when one tetranucleotide repeat, out of five or more markers studied, is mutated. Due to the lack of an animal model and low availability of human samples, EMAST studies have been limited and it has been difficult to determine exactly how EMAST relates to aggressive CRC. Our aim is to look at specific tetranucleotide repeats in mice to see if EMAST can be found. We are using tumors from a mouse model of colon cancer for our studies.
Human EMAST loci have been identified in tetranucleotide regions that show instability in population studies. But human EMAST loci are hard to identify in mice because humans and mice have less than 50% homology in non-coding DNA. Thus, bioinformatics was used to help identify mouse tetranucleotide repeats with potential for instability. Using a unique approach developed in our lab, mouse tetranucleotide repeats that were likely to be unstable were previously identified. For this project, several of these repeats were chosen for further study. Primers were developed, potential EMAST markers were amplified by PCR from normal mouse colon DNA, and sequenced. Once the sequence of the repeat was confirmed in normal tissue, those markers were checked for instability between normal and tumor tissues. To identify EMAST, we look for a gap in the repeat region between the normal colon and tumor DNA sequences that show there was a deletion or insertion of a tetranucleotide repeat between the tumor and normal DNA of the same mouse.
Of the ten potential markers we have evaluated so far, four display instability in normal DNA of individual mice, three display EMAST in tumors versus normal DNA, and five out of seven large and invasive tumors tested show EMAST in our mouse model of CRC. For the first time ever we have shown EMAST in an animal model of cancer. We will continue to test more EMAST loci and additional tumors to add to our panel of markers that can be used to study EMAST in any mouse model of cancer. This study is funded by NCI grant U01CA162147.
Poster #: 211
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: bacteria, toxin, probiotics
Project Title: Comparing potential cytotoxic effects of autotransporter toxin (Sat) in human uropathogenic E. coli (UPEC) and probiotic strain E. coli Nissle 1917
Author List:
Halsey, Taylor; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Abstract: Autotransporter proteins, typically associated with virulence factors in pathogenic bacteria, are thought to play a different role in the probiotic bacteria. One such protein, secreted autotransporter toxin (Sat), has been shown in numerous pathogenic bacteria to cause vacuolization and cytotoxic effects on cells. Sat is also secreted in high amounts by the non-pathogenic, probiotic Escherichia coli Nissle 1917 (EcN). EcN is being reviewed as a candidate probiotic supplement in the United States due to its beneficial effects on a host organism. The delays in approval by the Food and Drug Administration (FDA) are due to similarities to uropathogenic E. coli CFT073 (UPEC), a close genomic relative of EcN, which is responsible for approximately 70% of urinary tract infections nationwide. Sat of UPEC is known to play a role in cell detachment and death. Previous studies have indicated that when Sat functions collaboratively with other native proteins in EcN, its cytotoxic effects are negated but the mechanisms are unknown. To independently study the effects of Sat, we transformed a sat overexpression plasmid derived from UPEC (pSat) into the non-pathogenic E. coli strain HB101 (HB101/ pSat). An SDS-PAGE was performed to confirm the expression and secretion of Sat by HB101/ pSat. Next, we created a negative control strain by colony exclusion and antibiotic selection of HB101/ pdg4. The two strains were then used for in vitro infection studies using the HeLa cell line and analyzed with the MTT cytotoxicity assay. Our results show that Sat of UPEC damages the undifferentiated epithelial cells when subjected to infections longer than six hours. This suggests that Sat of UPEC works in a time-dependent manner to damage human epithelial cells. Further insight into the potential cytotoxic effects of Sat are needed to approve EcN as a probiotic supplement in the United States.
Poster #: 212
Campus: CSU Northridge
Poster Category: Disease (Pathogens)
Keywords: Toxoplasma gondii , IMC Proteins, CRIPSR
Project Title: Localization and Function of novel Toxoplasma gondii proteins
Author List:
Negrete, Pedro; Undergraduate, Biology, California State University, Northridge, Presenting Author
Malone, Cindy; BIOLOGY, California State University, Northridge
Bradley, Peter; UCLA
Abstract: Toxoplasma gondii is an obligate intracellular parasite that can infect all mammals and infects 1/3 of the human population. The primary methods of obtaining this parasite is through consumption of infected undercooked meat or infected cat feces through contaminated vegetables, meats, etc. Infection is lifelong and chronic, but usually asymptomatic in healthy individuals, although severe or, even fatal disease can occur in immunocompromised individuals. Primary maternal infection can cause severe birth defects. Encephalitis and chorioretinitis are common syndromes caused by Toxoplasma infection in adults and unborn children. The inner membrane complex (IMC) plays an important role in host cell invasion, parasite replication, and motility, all of which contribute to human disease. The IMC of Toxoplasma gondii lies just underneath the plasma membrane, supported on a network of intermediate filaments and microtubules. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. The purpose of this project is to localize IMC proteins associated with the IMC. We accomplished this by localization through endogenous gene tagging. We were able to successfully localize six proteins, three of which were either IMC or IMC associated. Gene 225940 was localized to the plasma membrane, gene 266080 was in the apical cap, and gene 231160 was in the IMC. Once we localized our IMC/ IMC associated proteins, we performed a western blot to see if our proteins would coincide with expected size based on the open reading frame. We discovered that the IMC and the plasma membrane proteins were larger than expected. We believe this could have been due to post translation modifications. For the apical cap protein, it was smaller, about 65 kDa, rather than the expected 86 kDa. We believe this was due to proteolytic cleavage which has been seen in other IMC proteins. Next, we performed CRISPR on these in order to knockout our gene of interest and look for potential phenotypes. We found that gene 231160 showed a distinguishable phenotype, namely death. This gene must be essential to the viability of the parasite. The other two genes did not show an obvious phenotype. For future experiments, we will assess the effects of gene 231160 with respect to overall growth and organelle inheritance in vitro, complement the mutant with the wild-type gene, and evaluate whether disruption affects virulence in vivo (in mice).
Poster #: 213
Campus: CSU Los Angeles
Poster Category: Disease (Pathogens)
Keywords: Innate immunity, Biofilm, Mycobacterium
Project Title: Establishing a Biofilm Assay with Mycobacterium smegmatis
Author List:
Berton, Roger; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Setayesh, Maryam; Graduate, Biological Sciences, California State University, Los Angeles
Mendez, Marlon; Undergraduate, Biological Sciences, California State University, Los Angeles
Porter, Edith; Biological Sciences, California State University, Los Angeles
Abstract: Background: Mycobacterium tuberculosis (Mtb) is the infectious agent responsible for tuberculosis (TB) that causes over 9 million illnesses annually. Multidrug resistance of Mtb and lack of a protective vaccine in the adult pose a major threat to the public. Our laboratory is interested in exploring whether host derived antimicrobial lipids may offer novel therapeutic approaches and vaccine development for TB. There is increasing evidence that biofilm production by Mtb may contribute to the infection process in vivo. Mycobacterium smegmatis (Ms) is often used as a model organism for Mtb because of its reduced pathogenicity and faster growth. A challenge working with Ms is it tendency to grow in aggregates precluding reliable colony forming unit assays. Objective: Establish biofilm assay with Ms to assess activity of host derived antimicrobial lipids. Methods: Isolated colonies of Ms were inoculated in 7H9 medium, grown for 24 – 96 h in 7H9 medium at 37 degree C, subjected to sonication to disperse bacterial aggregates, further diluted in 7H9 medium at varying concentrations, and inoculated in 96 well round bottom plates. Biofilm was quantified after 24 – 72 h according to standard crystal violet staining protocol. Results: We found that the most biofilm was produced with a 48 h culture diluted 1:50 and further incubated for 48 h and that replicates of 5 are necessary to accommodate remaining inhomogeneity after sonication. We are now in the process to assess the anti-biofilm activity of host derived lipids alone and in synergism with antimicrobial peptides. Conclusion: This research will generate a better understanding of how to inhibit mycobacterial biofilm production and may lead to novel drugs against Mtb. Acknowledgements: NIH 1SC1GM096916.
Poster #: 214
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: aspergillosis, vaccine, liposomes
Project Title: Efficacy of a Liposomal Aspergillus Vaccine to Provide Protection Against Mouse and Chicken Pulmonary Aspergillosis
Author List:
Reza, Hernan; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Olson, Jon; Biological Sciences, California State Polytechnic University, Pomona
Griggs, Lisa; Western University of Health Sciences, College of Veterinary Medicine
McNamara, Tracey; Western University of Health Sciences, College of Veterinary Medicine
Ho, Sam; Molecular Express Inc., Biological Sciences, California State Polytechnic University, Pomona
Adler-Moore, Jill; Biological Sciences, California State Polytechnic University, Pomona
Abstract: Introduction:Life-threatening pulmonary aspergillosis, caused by Aspergillus fumigatus, is most common in immunocompromised animals and avian species. Antifungal drug treatment is associated with only 50% survival, underscoring the need for an Aspergillus vaccine for people and birds at high risk for infection. There is also concern that this fungus is becoming resistant to the most widely-used drugs. This study was done to test the efficacy against pulmonary aspergillosis of a liposomal Aspergillus vaccine (LAsV) containing different adjuvants in immunocompromised mice and chickens.
Methods:In the mouse study, mice were vaccinated(n=17/gp, 5-6 wks old) d0 subcutaneously (sc),d21 and d42 intranasally (IN) with LAsV at 5ug Aspf3/dose and 5ug Aspf9/dose with the adjuvant 15ug monophosphoryl lipid A (MPL), 5ug lipidated Tucaresol (LT1), 25ug PAM3CAG, or 50ug dsRNA;controls were MPL liposomes or buffer. d53, d55, d57 mice were immunosuppressed with triamcinolone acetonide (28mg/kg) and d56 challenged IN with 1.5x10ex7 A. fumigatus. Lungs, BAL and blood were collected d59(n=6-7/gp)for fungal burden and serum anti-Aspf3 or Aspf9 IgG levels; remaining mice (n=9-10/gp) were monitored for morbidity to d77. SPF chickens (n=7/gp, 10 days old) were vaccinated d0 sc, d11 and d32 mucosally with LAsV at 5ug Aspf3/dose and 5ug Aspf9/dose with 15ug MPL or 6ug LT1/dose; control was buffer. d38, d39, d40, d41 chickens were immunosuppressed with dexamethasone (4mg/kg) and challenged d39 mucosally with 4x10ex8 A. fumigatus. Lungs and tracheas were collected from all chickens d46 and analyzed for fungal burden.
Results:In mice, LAsV with MPL or LT1 produced 70% and 60% survival, respectively, which was better than with the other adjuvants in LAsV, and significantly better than MPL liposomes (0%) (p≤0.011). ELISA serum IgG isotyping showed that LAsV with MPL elicited a Th1 response and LAsV with LT1 elicited a Th2 response. In chickens, LAsV with MPL or LT1 significantly reduced the fungal burden in the tracheas versus the control group (p≤0.048).
Conclusions:LAsV vaccines containing MPL or LT1 adjuvants generated significant protection against pulmonary aspergillosis in both mice and chickens. The adjuvants activated the immune responses by different pathways, with MPL stimulating a Th1 response and LT1 stimulating a Th2 response. This indicated that more than one immune pathway could be effective at generating protection against this fungal infection.
Poster #: 215
Campus: Cal Poly San Luis Obispo
Poster Category: Disease (Pathogens)
Keywords: Catheter-associated urinary tract infections, Urease, Polymicrobial infection
Project Title: Proteomic Analysis Identifies Proteins Associated with Synergistic Urease Activity in Polymicrobial Catheter-Associated Urinary Tract Infections
Author List:
McCready Reynolds, Shawn; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Mier, Joshua; Graduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Tomanek, Lars; Biological Sciences, California Polytechnic State University, San Luis Obispo
Yep, Alejandra; Biological Sciences, California Polytechnic State University, San Luis Obispo
Abstract: Catheter-associated urinary tract infections (CAUTI) account for approximately 40% of nosocomial infections, and 86% are polymicrobial. Gram-negative, urease-producing bacteria such as Proteus mirabilis and Providencia stuartii utilize urea present in urine as a nitrogen source, breaking it down into ammonia and carbon dioxide. This increases urine pH causing precipitation of Mg2+ and Ca2+ salts into crystalline structures (urinary stones). This may cause catheter blockage, and also increase bladder epithelium damage, which can result in bacteremia. Both in vivo and in vitro studies in coinfection or coculture show a synergistic increase in urease activity compared to single species, but the mechanism behind this synergy is unknown. The specific aim of this project is to identify protein(s), using tandem mass spectrometry (MS/MS), which are differentially expressed in co-culture of P. mirabilis and P. stuartii, compared to single species cultures, to uncover pathway(s) behind the observed urease synergy. Samples of individual and co-cultures of P. mirabilis and P. stuartii in pooled human urine were collected at time points 0, 30, 60, and 90 minutes to determine urease activity. Samples at 0 and 90 minutes were also collected for proteomic analysis. Bacterial cell lysates were processed for protein isolation. Quantified protein solutions were subsequently separated using 2D gel electrophoresis and analyzed using DELTA-2D (DECODON) to determine changes in protein abundance. 20 spots exhibited significant changes in protein abundance in response to both time and coculture (two-way ANOVA, p < 0.05). All protein spots were extracted and processed for identification by MS/MS. Out of the 20 significant proteins, 9 have been identified: dihydrolipoyl dehydrogenase, D-alanyl-D-alanine carboxypeptidase, bifunctional metallophosphatase/5′ nucleotidase, Class II fumarate hydratase, elongation factor G, outer membrane channel protein TolC, DNA-directed RNA polymerase subunit alpha, porin, and phosphoenolpyruvate carboxykinase. Most of the differentially abundant proteins derive from P. mirabilis, suggesting that the synergistic urease activity results from a P. stuartii stimulus acting on P. mirabilis. We are currently working towards generating genomic mutants to identify the role of these proteins in urease synergy, as well as identifying the remaining proteins. This work was supported by a Cal Poly COSAM Frost fellowship (SMR) and start-up funds (AY).
Poster #: 216
Campus: San José State University
Poster Category: Disease (Pathogens)
Keywords: inflammation, proprioception, muscle
Project Title: Changes in muscle sensory afferent response to mechanical stimulation following lipopolysaccharide-induced systemic inflammation
Author List:
Zaytseva, Dasha; Undergraduate, Biological Sciences, San José State University, Presenting Author
Padmanabhan, Shreejit; Undergraduate, Biomedical Engineering, San José State University, Presenting Author
Criddle, Connor; Undergraduate, Chemistry, San José State University
Wilkinson, Katherine A.; Biological Sciences, San José State University
Abstract: Inflammation is linked to a variety of medical conditions, including chronic pain states and musculoskeletal disorders. Many of these inflammatory conditions are characterized by a higher prevalence in women. In addition, several studies have found significant sex differences in response to inflammation (Manson 2010). Inflammatory factors such as bradykinin and reactive oxygen species have been shown to alter the response of muscle sensory afferents to mechanical stimulation (Delliaux et al. 2009), which may contribute to the development of these conditions. We investigated sex differences in the response of muscle sensory afferents to physiological stretch and vibration following acute systemic inflammation. Inflammation was induced by an intraperitoneal injection of lipopolysaccharide (LPS; 7.5×10^5 EU/kg; control 200 µL saline) given 18 hours prior to the experiment. The extensor digitorum longus muscle and sciatic nerve of adult C57Bl/6 mice were dissected and placed in an in vitro bath perfused with synthetic interstitial fluid (SIF) and 100% oxygen. The nerve was suctioned into a microelectrode and muscle sensory afferent activity was recorded in response to a series of physiological ramp-and-hold stretches at 3 lengths (2.5%, 5%, and 7.5% of the resting length), and sinusoidal vibrations at 4 amplitudes and 4 frequencies (10, 25, 50, 100 mm; 10, 25, 50, 100 Hz). In addition, the series and parallel muscle elasticity was calculated and compared between treatment groups. Individually identified muscle stretch receptors showed no significant changes in response to stretch following inflammation, although there was a sex difference. However, male and female LPS animals displayed a similar reduction in ability to entrain to high frequency vibration, indicating a decrease in dynamic sensitivity of the muscle stretch receptor afferents. These findings suggest systemic inflammation causes altered proprioceptive information to be relayed to the central nervous system. We found no significant differences in the anatomical or passive muscle mechanical properties, including muscle elasticity, signifying the existence of an unknown underlying mechanism for inflammation-induced changes in afferent sensitivity.
Work was funded by CSUPERB Presidents’ Commission Scholars (DZ, SP), SJSU Provost’s Undergraduate Research Grant (DZ, SP), & a CSUPERB New Investigators Grant (KAW).
Poster #: 217
Campus: CSU Fullerton
Poster Category: Disease (Pathogens)
Keywords: Ion channels, calcium, electrophysiology
Project Title: Physiological role of a Calcium Activated Potassium Channel in Trypanosoma cruzi
Author List:
Skorka, Christopher ; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Boktor, Michael; Undergraduate, Biological Science, California State University, Fullerton
Jimenez, Veronica; Biological Science, California State University, Fullerton
Abstract: Chagas Disease is a parasitic infection caused by the protozoan Trypanosoma cruzi. The parasite faces various environmental changes as it develops in an insect vector and mammalian hosts. Osmolarity, ionic concentrations and pH are highly variable throughout the parasite’s life cycle, and is the ability to adapt and respond to such conditions what determines the survival and successful transmission of T. cruzi.
We propose that different types of channels integrate a homeostatic network that allows the parasite to detect and respond to environmental changes. Combining molecular, cellular and electrophysiological approaches we are characterizing the expression and function of a putative calcium-activated potassium channel TcCAKC. By immunofluorescence, we have localized the channel in the plasma membrane of the three main developmental stages of the parasite. In order to characterize the biophysical properties of the channel, we have expressed TcCAKC in Xenopus laevis oocytes by cRNA microinjection followed by two-electrode voltage clamp.
Oocytes expressing TcCAKC and exposed to voltage pulses show a significant increase in inward currents after addition of ionomycin, a calcium ionophore. Similar responses can be elicited by pre-incubation of the cells with thapsigargin, a SERCA pump inhibitor that increases free cytosolic calcium. The responses are abolished by addition of EGTA, suggesting that TcCAKC activation depends on extracellular calcium. Ion selectivity, conductance and inactivation mechanisms are under currently under study.
We were able to generate parasite cell lines in which one (knockdown) or both alleles of the gene (knockout) have been eliminated. The non-infective forms of the parasite (epimastigotes) do not have a growth defect, but they show a significant decrease in their ability to infect mammalian cells.
To pinpoint at the mechanism by which TcCAKC could be regulating the infectivity of the parasites, we measured membrane potential (Vm) of the parasites using the fluorescent probe DisBac2 (3). TcCaKC knockouts were much more hyperpolarized than knockdowns or wild type parasites, suggesting that the activity of the channel could be related with the maintenance of the membrane potential, and indirectly regulating the calcium dynamics necessary for transformation of the parasites into infective forms and successful invasion of host cells.
Funding: NIH-NIAID R00AI101167
Poster #: 218
Campus: San Francisco State University
Poster Category: Disease (Pathogens)
Keywords:
Project Title: 3D reconstruction of the Locus Coeruleus of Early Stages of Alzheimer’s Disease as a Visualization Tool for Progression Monitoring.
Author List:
Castruita, Patricia; Undergraduate, 2015 University of California San Francisco Summer Research Training Program, San Francisco State University, Presenting Author
Theofilas, Ph.D., Panos; Department of Neurology, University of California San Francisco
Ribeiro, Ph.D., Maryana; Department of Neurology, University of California San Francisco
Grinberg, MD, Ph.D., Lea T.; Department of Neurology, University of California San Francisco
Abstract: Recent studies in Alzheimer’s disease (AD) performed by our research team have identified a volumetric decline in a specific subcortical region responsible for norepinephrine synthesis in the brain, the Locus Coeruleus (LC). Such changes, not typical of normal aging, reported to happen during the early stages of AD prior to neuronal loss. As a proof of principle, we performed a three dimensional reconstruction of three brainstems obtained from the Neurodegenerative Disease Brain Bank (NDBB) at the University of California San Francisco, all cases manifesting early stages of AD pathology (Braak 0-3). The novelty of our study is the use of a pipeline that combines block face images and histological segmentation along with high resolution 2D imaging allowing for precise representation of subcortical structures using an in- vivo view of the brainstem compared to previously represented examples. The Brainstem was sectioned, nissl stained, and processed for imaging prior to reconstruction. Utilizing high resolution microscopy imaging along with 3D data visualization software (AMIRA, Konrad-Zuse-Zentrum), each case was segmented and aligned in ascending order for reconstruction while the region of interest (LC) was manually traced for better identification. Our brainstem reconstructions provide optimal visualization with an end product of qualitative data which can be used in the future for comparative analysis to magnetic resonance imaging scan reconstructions.
Poster #: 219
Campus: San Diego State University
Poster Category: Disease (Pathogens)
Keywords:
Project Title: The cell surface adhesin BspC contributes to Group B Streptococcal meningitis
Author List:
Vant Hul, Katilynne N.; Graduate, Department of Cell and Molecular Biology, San Diego State University, Presenting Author
Deng, Liwen; Graduate, Department of Cell and Molecular Biology, San Diego State University, Presenting Author
Nobbs, Angela H.; Undergraduate, Department of Cell and Molecular Biology, San Diego State University
Doran, Kelly S.; Department of Cell and Molecular Biology, San Diego State University
Abstract: Streptococcus agalactiae (Group B Streptococcus, GBS) is a Gram-positive bacterium typically found within the human gastrointestinal and urogenital tract. GBS is currently the leading cause of neonatal meningitis due to its multiple virulence factors. In order to gain access to the central nervous system, GBS must penetrate the blood-brain barrier (BBB), which is composed of specialized cells known as human brain microvascular endothelial cells (hBMEC). The mechanisms of the BBB crossing are not well understood. We hypothesized that a member of the Antigen I/II family of cell surface anchored proteins, BspC, may promote GBS interaction with brain endothelium. Antigen I/II family proteins are known virulence factors that promote colonization of the oral cavity by other streptococci. To assess whether BspC contributes to GBS-BBB interaction, we performed allelic replacement of the bspC gene. Scanning electron microscopy revealed that the ΔbspC mutant exhibited altered surface appearance and decreased ability to interact with neighboring cells. To examine the role of BspC in the interaction of GBS with host cells we infected hBMEC with wild-type (WT) and the ΔbspC mutant and found that the mutant exhibited reduced ability to interact with endothelium. Further, using a murine model of hematogenous meningitis, we observed that mice challenged with the ΔbspC mutant exhibited a significant decrease in mortality and less bacterial brain loads compared to WT infected mice. These results suggest that BspC promotes virulence and BBB passage. Ongoing studies are underway to determine the exact mechanism of how BspC contributes to the pathogenesis of GBS meningitis.
Poster #: 220
Campus: Cal Poly San Luis Obispo
Poster Category: Disease (Pathogens)
Keywords: viable-but-nonculturable, Vibrio parahaemolyticus, flow cytometry
Project Title: Optimizing Methods for the Detection and Enumeration of Vibrio parahaemolyticus, and for the Induction of Cells into a Viable but Not Culturable State
Author List:
Miller, Michael; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Yeung, Marie; Biological Sciences, California Polytechnic State University, San Luis Obispo
Abstract: Vibrio parahaemolyticus is one of the primary pathogens causing foodborne illness in US, and has had a 32% increase in incidence from 2006 to 2013. A potential cause of this increase of incidences is the bacterium’s enhanced virulence and/or its ability to persist in an environment while being difficult to be detected due to decreased metabolic activity. Pathogenic cells that are in a viable but not culturable (VBNC) state are overlooked by typical culture-dependent methods. Thus, other methods are necessary to study VBNC cells. In addition, it is important to harvest sufficient amount of VBNC cells to study their physiology; however, it is a time-consuming process as previous studies showed that it took months to yield VBNC cells in laboratory conditions. The objectives of this study were to evaluate methods for the detection and enumeration of Vibrio parahaemolyticus cells that are in the VBNC state; and to develop a procedure to induce cells to enter VBNC state in a more controlled setting. By using a concentrations of ~1e8 cells/ml live and dead cells, 1:10 serial dilution was made. Comparisons among several methods – naladixic acid assay, ATP bioluminescence, fluorescence microscopy, flow cytometry – were conducted to enumerate and differentiate viable and nonviable cells. Standard plate count was done in conjunction with all methods to determine the culturablility. The best method was found to be flow cytometry (533 and 670 nm) coupled with fluorophores SYTO9 (5 µM) and propidium iodine (20 µM) because it achieved ≥99% sensitivity and ≥95% specificity; and all readings were within the expected cell number ranges. Next, four media types – Modified Morita’s mineral salt solution (MMS), artificial seawater, natural seawater, tryptic soy broth with salt (TSBS) – and three incubation temperatures (5, 22, 37 degree Celsius) were evaluated to determine which combination would yield the highest percentage of VBNC cells in the shortest amount of time. Incubation in MMS at 5 degree Celsius for 3 weeks yielded ≥99.9% of VBNC Vibrio parahaemolyticus cells. In conclusion, we successfully prepared VBNC cells and developed a sensitive method to quantify them. Our next step is to study the effects of VBNC on the pathogenicity of Vibrio parahaemolyticus. Funding is partly supported by Graduate Women in Science.
Poster #: 221
Campus: CSU San Bernardino
Poster Category: Disease (Pathogens)
Keywords: Traumatic brain injury , D. melanogaster , Alzheimer’s Disease
Project Title: The Use of Drosophila melanogaster as a model to Investigate the Underlying Cellular and Molecular Basis of TBI
Author List:
Silva, Ivan ; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Rai, Taj; Undergraduate, Chemistry and Biochemistry, California State University, San Bernardino, Presenting Author
Medina, Pedro; Undergraduate, Biology, California State University, San Bernardino
Bournias-Vardiabasis, Nicole ; Biology, California State University, San Bernardino
Abstract: Each year in the US., 1.7 million people experience a traumatic brain injury (TBI). A TBI is characterized by the severe impact to the cranial region in which the brain consequently slams against the inside of the skull. This can result in swelling and bruising of the brain, which can lead to decreased brain function. TBI is a significant health issue worldwide, yet the mechanism responsible for its complex pathologies remains largely unknown. Sustained TBI’s have been shown to increase the likelyhood of Alzheimer’s (AD) and other neurodegenerative diseases. Our research aims to identify some of the possible cellular and molecular mechanisms of TBI by utilizing the model organism D. melanogaster. Two strains of D. melanogaster were used in this study, a transgenic Aβ 42 strain that expresses a 42 amino acid long peptide and a genetically matched 00C strain that served as the control. The accumulation of amyloid-β (Aβ)42 peptide has been hypothesized to be the primary event in AD pathogenesis. The Drosophila TBI model consists of the use of a spring action apparatus that delivers a strong jolt of 0.1 sec. The experimental design included: 00C flies that received 0, 5 or 10 daily TBI’s, and Aβ-42 flies, Aβ -42 that received 0, 5 or 10 daily TBI’s . Lifespan, cognitive ability (negative-geotaxis assay), cholinergic neuron viability as assessed by GFP expression, and cholinergic neuron quantity were analyzed to assess the effects of TBI. Although it was found that both Aβ-42 and the control showed the same inverse relationship between TBI treatments and lifespan, the Aβ-42 strain experienced a shorter lifespan than the control. The climbing assay also showed the same trend for both strains in that increased daily TBI inducing treatments resulted in decreased climbing ability, however, each treatment group in the Aβ-42 strain showed a greater decreased climbing ability than that of the 00C strain. In terms of cholinergic neuron function, groups treated with ten daily TBI’s experienced the sharpest decline. Neuronal function assessment indicates the Aβ-42 strain had a lower average and lower deviation in measured intensity of GFP expression, whereas the control had a higher average and higher deviation in measured intensity of GFP expression. In future studies, behavioral assessments could also be incorporated such as a sugar preference assay or odor avoidance assay as they might give us a better insight on the behavioral pathologies identified in humans.
Poster #: 222
Campus: Sacramento State University
Poster Category: Disease (Pathogens)
Keywords: Acne, Metabolite, Lipogenesis
Project Title: Propionibacterium acnes Alters Lipid Synthesis by Human Sebocytes
Author List:
Singh, Shaleni; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Davis, Tyler; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Crawford, Robert; Biological Sciences, California State University, Sacramento
Abstract: Inflammatory infections of the lipid-rich pilosebaceous gland attributed to the Gram positive bacterium Propionibacterium acnes are largely uncharacterized and understudied due to the paucity of anaerobic methods culture methods and the contamination of clinical samples by species of the resident skin microbiota. Infections are often attributed as absent due to subtleties in symptom manifestations, particularly for patients with indwelling medical devices or post-surgery prosthetics. A renewed interest in studying the role of P. acnes in health and dysbiosis has emerged thanks in large part to the identification of transcriptomic and phylogenetic signatures generated by whole genomic sequencing data from the Human Microbiome Project. We hypothesize here that P. acnes regulate lipogenesis during acne to remodel the local microbiome through altered metabolite production. In what is the first experiment in the field to co-culture primary pilosebaceous cells and live bacteria, Western blot data from our lab shows that human sebocytes obtained from human donors produce significantly more of the lipogenesis regulatory factor mechanistic target of rapamycin (mTOR) following two or four hour exposures to P. acnes relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in uninoculated controls. Similar results were obtained for other probes in the lipid synthesis pathway including protein kinase B (AKT). To confirm these findings, we performed AdipoRed analysis and show that lipogenesis is in fact significantly increased when sebocytes are cultured with live P. acnes relative to unstimulated controls, heat killed bacteria, or filtered bacteria supernatants. Interestingly, RNA sequencing analysis showed that bacteria exposed to sebocytes have an altered transcriptome, and metabolomics screening following principal coordinate analysis of co-culture supernatants revealed an altered metabolite profile compared to organisms grown in isolated broth cultures. We are currently isolating and purifying these metabolites produced during P. acnes co-culture with sebocytes and testing their effects on the growth and biofilm formation of other skin-associated bacteria including Staphylococcus epidermidis. These studies represent collaborative efforts between UC Davis and CSUS and were supported by the Albert Delisle Scholarship for undergraduate research.
Poster #: 223
Campus: San José State University
Poster Category: Disease (Pathogens)
Keywords: sensory neurophysiology, exercise, proprioception
Project Title: Local Effect of Reactive Oxygen Species on Muscle Spindle Afferent Function in Adult Male Mice
Author List:
Kwon, Anthony; Graduate, Biological Sciences, San José State University, Presenting Author
Damani, Milind; Undergraduate, Biological Sciences, San José State University, Presenting Author
Wilkinson, Katherine; Biological Sciences, San José State University
Abstract: Muscle spindle group Ia and II afferents relay information about changes in muscle length and allow one to determine body position and movement in space. Impairments in muscle spindle afferent (MSA) function are linked to an increased risk of falling, reduced balance, and sport-related injury. Exercise-induced fatigue has been shown to alter MSA function, likely due to exercised-released metabolites, including reactive oxygen species (ROS). However, the mechanism of how exercise-released metabolites affect receptor endings of muscle spindle afferents in the periphery has yet to be characterized. We tested the hypothesis that ROS can act locally on muscle spindle afferent receptor endings and modify responsiveness to stretch and high frequency vibrations. The extensor digitorum longus (EDL) muscle and innervating sciatic nerve were dissected and placed into an in vitro tissue bath of oxygenated synthetic interstitial fluid. MSA static stretch response was determined by subjecting the EDL to 4s ramp and hold stretches at a single physiological stretch length (5% of resting length) and measuring afferent instantaneous firing frequency via extracellular recording of the sciatic nerve. MSA dynamic sensitivity was determined by whether MSA could entrain to high frequency vibrations (50 µm amplitude, 50 Hz and 100 Hz). Muscle tension was measured before and throughout ramp and hold stretches and high frequency vibrations. Ramp and hold stretches and high frequency vibrations were then repeated in the presence of ROS (1 mM hydrogen peroxide). Hydrogen peroxide exposure resulted in an increased static stretch response in about half of the MSAs tested and did not alter the activity of the other half. We also observed increased muscle tension during ramp and hold stretches during hydrogen peroxide exposure. High frequency vibration entrainment data is still being analyzed. These results support our hypothesis that hydrogen peroxide can act locally on muscle spindle afferent receptor endings and modify responsiveness to stretch. Specifically, our results reflect the existence a subpopulation of MSA that are sensitive to hydrogen peroxide and possibly other reactive oxygen species. Future studies will investigate changes in MSA static stretch response and dynamic sensitivity following fatiguing contractions.
Poster #: 224
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: alternative splicing, RNA binding proteins, cloning
Project Title: Identification and Characterization of a Minimal Functional Splicing Regulatory Protein
Author List:
Ontiveros, Robert; Undergraduate, Biological Sciences, California State University, Fullerton, Presenting Author
Doan, Justin; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Keppetipola, Niroshika; Chemistry and Biochemistry, California State University, Fullerton
Abstract: In higher eukaryotes, alternative splicing of a single gene transcript into multiple final spliced mRNA contributes significantly towards the diversity of cellular proteins. The process of alternative splicing is regulated in part by RNA binding proteins that bind to RNA adjacent to regulated exons and influence the assembly of a functional spliceosome at adjacent splice sites. Aberrant alternative splicing has been identified in many diseases such as Alzheimer’s disease, muscular dystrophy, and ovarian cancer, underscoring the importance of alternative splicing. Detailed mechanistic and molecular understanding of how splice variants are generated will provide new targets for therapeutic interventions. The Polypyrimidine Tract Binding Protein 1 (PTBP1) is a well characterized RNA binding protein with roles in alternative splicing regulation, mRNA localization, and IRES-mediated translation initiation. PTBP1 binds preferentially to either upstream or downstream sites of target exons to promote their exclusion or inclusion from the spliced transcript. PTBP1 contains four RNA Recognition Motifs (RRMs) which each bind to pyrimidine rich sequences with varying affinity and structural preferences and are joined by three linker peptides. The complete atomic structure of PTBP1 bound to a target RNA and the interaction(s) between RRM’s while bound to RNA is unknown. Attempts to crystallize RNA-bound PTBP1 have been hindered by the flexible linker regions between RRM1 and 2 (linker 1, 42 aa’s) and RRM 2 and 3 (linker 2, 81 aa’s). In this study, we aim to identify and characterize a functional minimal-linker PTBP1 mutant for structure studies via x-ray crystallography. To this end, a series of PTBP1 mutants were generated using 2-step PCR with deletions in both linker 1 (Δ19, Δ29) and 2 regions (Δ21, Δ44, Δ53, Δ68). Mutants were tested for protein expression and splicing repression activity in vivo. Western blots indicated that each mutant is well expressed. Splicing assays on three PTBP1-regulated test exons reveal that the minimal construct (PTBP1 L1Δ40 – L2Δ68) maintains splicing repression activity comparable to full-length PTBP1. We have sub cloned PTBP1 L1Δ40 – L2Δ68 into an E. coli expression vector and performed a large-scale protein purification. Purified protein samples indicate that in vitro expressed PTBP1 L1Δ40 – L2Δ68 is soluble and can be purified for use in x-ray crystallography.
Poster #: 225
Campus: Humboldt State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Lgl, cJun, JNK
Project Title: Loss of Lethal Giant Larvae Lgl1 Increases c-Jun Expression and Phosphorylation in Murine Neural Progenitor Cells
Author List:
Otis, Sharon; Graduate, Biological Sciences, Humboldt State University, Presenting Author
Petersen, Abigail ; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
Trzeciak, Jacqueline; Undergraduate, Biological Sciences, Humboldt State University
Collins, Hannah; Undergraduate, Biological Sciences, Humboldt State University
Sprowles, Amy; Biological Sciences, Humboldt State University
Abstract: High grade gliomas are aggressive and highly invasive brain cancers which harbor stem cell like tumor propagating cells and originate from neural stem/progenitor cells in genetically engineered mouse models (Singh et al. 2004, Llaguno et al. 2009). Aberrant activities of the tumor suppressor Lethal Giant Larvae homolog 1 (LGL1), c-Jun N-terminal kinase (JNK), and the protooncogene cJun are associated with high grade glioma and each have independently been shown regulate tumorigenic and/or malignant properties in mouse models or glioma cell lines (Klezovitch et al. 2004, Mangiola et al. 2007, Gont et al. 2013, Yoon et al. 2012, Blau et al. 2012). LGL regulates cell polarity and the actin cytoskeleton. It has been shown to suppress Jun-N terminal kinase (JNK) activity in Drosophila (Sun and Irvine 2011) and changes in cytoskeletal dynamics have been shown to increase translation of c-jun (Pollack 2006). We predicted the loss of LGL would increase JNK and cJun activity in neural progenitor cells by JNK signaling, cytoskeletal disruption, or both. Neuroprogenitor cells were isolated from the subventricular zone (SVZ) of two different neonatal Lgl1 loxp/loxp mouse brains and treated with AD-CRE GFP or AD-GFP. Analysis by Western blot and immunocytochemistry shows increased nuclear cJun in both Lgl1 -/- and Lgl1 +/+ cell lines. The cells were then treated with DMSO, 10 ng/ml anisomycin, or 5 ug/ml nocodazole. Our results show cJun is more phosphorylated in Lgl-/- cells 1 hour post anisomycin treatment. The phosphorylation is inhibited by SP600125, indicating JNK is the phosphorylating kinase. Treatment with DMS0 or nocodazole increased cJun expression in both cell lines within 1 hour, but Lgl -/- cells have increased stress fiber formation, increased viability and significantly more cJun 48 hours post treatment. We conclude loss of Lgl1 does increase JNK activity, but that the increased expression of cJun is likely do to changes in translational control by the cytoskeletal network. Additional studies will be required to understand if these mechanisms are involved in tumor formation and differentiation of neural progenitor cells.
Poster #: 226
Campus: Sacramento State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Acinetobacter baylyi, Microbial Genetics, Gene Amplification
Project Title: Optimizing and Categorizing the selection for gene amplification mutants in Acinetobacter baylyi
Author List:
Sami, Josephine; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Reis, Elizabeth; Graduate, Biological Sciences, California State University, Sacramento
Quinones-Soto, Semarhy; Biological Sciences, California State University, Sacramento
Reams, Andrew; Biological Sciences, California State University, Sacramento
Abstract: Using the soil bacterium Acinetobacter baylyi, this study aims to optimize a model system to study the phenomenon of gene amplification. In this system, parent cells expressing only basal amounts of the genes involved in degrading benzoate (cat genes) are incapable of utilizing benzoate (Ben-) as its sole carbon source. However, rare spontaneous mutant colonies (Ben+) carrying cat gene amplification emerge over time on selective benzoate plates. While other groups have previously reported cat gene amplification mutant colonies accumulating at a rate of 1 per 109 parent cells in 14 days, we observed a 10-fold lower mutant accumulation rate of 1 per 1010 parent cells in 14 days. Our goal was to test variable conditions on the Ben+ reversion rates. We have tested variable selective media recipes, incubation temperatures, parent strain genotypes and cell densities to determine the optimal conditions where cat gene amplification mutants accumulate at the highest possible rate. Our results show reversion rates vary significantly based on the parent strain genotype and the pre-growth conditions. The highest reversion rates were found using pre-growth temperatures of 37 degrees Celsius and by using a parent strain lacking an operative constitutive mutation in the genes responsible for converting benzoate to catechol (ben genes). Independently isolated mutant colonies were purified and characterized by PCR and Pulse Field Gel Electrophoresis to verify and quantify the level of cat gene amplification. Furthermore, we have compared Luria-Delbrück fluctuation assays with regular reversion assays to test the theory that the stress of the selective media can induce gene amplification.
Poster #: 227
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: tm7, lactase, metagenome
Project Title: Expression of a Novel Lactase Gene Found in the Uncultivated Bacterium TM7
Author List:
Grabski, Paige; Undergraduate, Biology, San José State University, Presenting Author
Ghadiri, Farsheed; Graduate, Biology, San José State University, Presenting Author
Cross, Haley; Undergraduate, Biology, San José State University
Lee, Becky; Graduate, Biology, San José State University
Romero, Diana; Undergraduate, Biology, San José State University
Caldwell, Adam; Undergraduate, Biology, San José State University
Pham, David; Undergraduate, Biology, San José State University
Abstract: The human oral cavity harbors hundreds of distinct bacterial species. Diet, gender, overall health, hormone levels, and genetics- all influence the oral microbiota in different aspects. However, most oral species of bacteria are uncultivable, meaning they cannot currently be isolated in pure culture. Because of such restrictions, very little is known about the effects of oral bacteria on human oral health. A notable uncultured bacterium, Candidatus Saccharibacteria, commonly known as TM7, has been associated with the human oral disease periodontitis. Although TM7 genomes have been partially sequenced, and the potential metabolic function of its genes have been proposed in silico, no TM7 genes have been empirically validated for their function. Our goal was to further characterize the TM7 bacterium by validating its functional genome by measuring enzymatic activity of a novel metabolic gene. Our methods consisted of generating a fosmid (40kb insert) library using genomic DNA extracted from an environmental sample known to contain TM7 bacteria. The library consisted of 4,000 fosmids, all of which were sequenced using Illumina technology and annotated using multiple bioinformatics tools. Four TM7 fosmids were identified in the library, and fosmid TM7-1 harbored a hypothetical lactase protein. Gene annotation identified a potential novel TM7 lactase, which was subcloned into a PET28a expression vector, and transformed into E coli mutants without lactase (lac-). Initial tests of the TM7 lactase against various substrates confirmed its lactase hydrolysis function. We then tested TM7-lac+ strains for its optimal conditions (temperature, pH, substrate concentration) and specific activity by means of ONPG (ortho-Nitrophenyl-β-galactosidase) assays. The size of the TM7 lactase was ~ 66kDa, half the size of an E.c. homologue. For substrate specificity we used various lactose analogs. These assays form a crucial first-step towards determining the metabolic capabilities of the uncultivated TM7. We have now a better understanding of the potential environment conditions to which the TM7 lactase evolved for optimal activity and its breath of substrates. By gaining a better understanding of the uncultured bacterium’s metabolism, we can potentially create a method for cultivating it in a laboratory setting.
Poster #: 228
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: manganese, genetic system, transposon mutagenesis
Project Title: Development of a Genetic System for the Mn(II)-oxidizing Erythrobacter sp. SD21 for Transposon Mutagenesis and Inducible Protein Expression
Author List:
Hsu, David; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Garoian, Alec; Undergraduate, Biological Science, California State University, Fullerton
Johnson, Hope; Biological Science, California State University, Fullerton
Abstract: Manganese (Mn) is an essential element that is needed for many biological processes and is involved in the biogeochemical cycling of many elements. Mn oxides acts as a sorbent or oxidant for various toxic metals which makes it ideal for bioremediation. To be used in bioremediation, Mn(II) has to be oxidized, and Mn-oxidizing bacteria are the most efficient method. Erythrobacter sp. SD21 is unique to Mn(II)-oxidizing bacteria, since it uses a novel Mn(II)-oxidizing protein. To better study this novel protein, a genetic system was developed in Erythrobacter sp. SD21 that was tested against three broad host-range plasmids and the transposon carrying vector, pUT/mini-Tn5Km. After successful transformation by each of the plasmids was shown through both chemical and electroporation means, two applications of the genetic systems were explored. The first application was to study potential genes that regulate Mn(II)-oxidation in Erythrobacter sp. SD21 by creating a random mutant library using transposon mutagenesis. The transposon carrying vector, pUT/mini-Tn5Km, randomly inserts a transposon carrying kanamycin resistance gene into Erythrobacter sp. SD21’s genome. Transposon-carrying mutants that have a change in Mn(II)-oxidation will then be analyzed through inverse PCR and sequencing to determine which gene is affected by the insertion. So far, five loss of function mutants have been isolated. The second application of the genetic system was to clone an inducible form of the novel Mn(II)-oxidizing protein into pJN105, the broad host-range plasmid with the highest transformation efficiency (1.3 x 10^5 cfu/µg). This was performed using the Polymerase Incomplete Primer Extension (PIPE) cloning method. Current and continuing studies include production of additional transposon mutants, analysis of transposon insertion site for mutants affected in Mn oxidation, and expression of the recombinant protein in the native host. Acknowledgements: This research is made possible by the Fuqua Lab at Indiana University Bloomington for their gifts of plasmids, the Tolmasky Lab at California State University Fullerton for the use of their electroporator, and the Associated Students, CSUF, Inc., and CSUPERB for funding.
Poster #: 229
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: zebrafish, stem cells, blood
Project Title: Ism-1 depletion negatively affects vertebrate hematopoiesis
Author List:
Berrun, Arturo; Graduate, Department of Biological Sciences, California State University, Chico, Presenting Author
Laurie, Payton; Graduate, Department of Biological Sciences, California State University, Chico, Presenting Author
Stachura, David; Department of Biological Sciences, California State University, Chico
Abstract: Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. The molecular processes that cause HSPCs to proliferate and differentiate into particular cell lineages aren’t fully understood. Hematopoiesis is a conserved evolutionary process among vertebrates, allowing the use of model organisms such as the zebrafish (Danio rerio) to investigate these processes. We generated and investigated three hematopoietic-supportive stromal cell lines derived from different sites of zebrafish blood production and maintenance, which encourage the growth and differentiation of HSPCs. RNA sequencing analysis indicated 447 genes that were shared among these cell lines that were not expressed in non-hematopoietic supportive zebrafish stromal cell lines. A highly expressed transcript in all of these cell lines was ism-1, a secreted protein that is involved in angiogenesis. In order to identify the role of ism-1 in hematopoiesis, we performed loss-of-function experiments by injecting morpholinos directed against ism-1 into the one-cell-stage zebrafish embryo. Utilizing transgenic zebrafish that have fluorescent myeloid and erythroid cells, we saw that ism-1 depletion reduced the number of myeloid cells and negatively affected blood circulation when analyzed by flow cytometry and fluorescent microscopy. We also enumerated hematopoietic progenitors in vitro, which were reduced in morphant embryos. These results indicate that ism-1 is an important gene in normal vertebrate hematopoiesis; future studies are aimed at determining if overexpressing this gene will be able to generate more blood in zebrafish. Identifying and chemically modulating transcripts involved in making blood can be utilized clinically, allowing the expansion of blood stem cells in culture to treat a multitude of diseases such as anemia and leukemia.
Poster #: 230
Campus: San Diego State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: whole genome sequencing, phenotypic microarray, bioinformatics
Project Title: Intersection of Genotype and Phenotype: Informing Predictive Models
Author List:
de Wardt, Rebecca; Graduate, Biology, San Diego State University, Presenting Author
Aguinaldo, Kristen; Graduate, Biology, San Diego State University
Cuevas, Daniel ; Graduate, Computer Science, San Diego State University
Lopez, Tucker ; Graduate, Public Health , San Diego State University
Cavalli, Dominic; Undergraduate, Biology, San Diego State University
Turnlund, Abigail ; Undergraduate, Biology, San Diego State University
Edwards, Robert; Computer Science, San Diego State University
Dinsdale, Elizabeth; Biology, San Diego State University
Abstract: Next generation sequencing has made whole genome sequencing an inexpensive and efficient way to analyze bacterial genomes. Genotypes offer many clues about phenotype, but all metabolic abilities of the organism may not be uncovered due to homology dependent annotations, limited databases and sequencing errors. Current phenotype studies are limited to a few traits. Our research applies a high-throughput approach to analyze 154 phenotypic traits of bacteria. Here, we start the analysis by comparing gene content of 15 marine bacteria and their phenotypic profiles for 71 carbon and 23 nitrogen sources. Isolated marine bacteria were individually cultured; whole genome sequencing was completed on each isolated bacterium using Illumina MiSeq. Genomes were annotated with Rapid Annotation using Subsystem Technology (RAST), which identified an average of 5,082 coding sequences. RAST suggests these bacteria are Vibrio or Shewanella spp. The genomes had an average of 1,419 hypothetical proteins, and our research could help identify these unknown genes. Analysis of genes present, using Bray Curtis clustering, showed the genomes divided into 3 major clades with 10 genomes sharing greater than 90 % similarity. Distance-based redundancy analysis (db-RDA) comparing gene content, demonstrated that hydroxymyristoyl-dehydratase, hydrogenase nickel incorporation-associated protein HypB, 1-4 dihydroxy-2-naphthoyl-CoA-hydrolase, and aclytransferase family protein attributed to 70.8 % total variation. However, when phenotypes of the bacteria were compared, a different clustering pattern occurred. Bray Curtis clustering of phenotypes showed only 80 % similarity for 11 bacteria with 2 major clades. The db-RDA revealed that carbon sources: pyruvate, salicoside and L-methionine and nitrogen source: beta-phenylethylamide, contributed to 71.9 % of total variation between bacteria. No compound tested was utilized as a sole carbon or nitrogen source by all the bacteria. Several carbon sources showed no growth, but at least one bacterium grew on all nitrogen sources. The discontinuity between phenotype and genotype, suggests that some of the hypothetical genes could be contributing to the phenotypes we describe. The data has already improved predictive growth models and will aide in bioprospecting efforts to identify commercially useful microbes.
Poster #: 231
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: pertussis, innate lymphoid cells ILC3, immune response, ,
Project Title: The role of Type 3 Innate Lymphoid Cell (ILC3) and Interleukin-23 secreting Dendritic Cells during Bordetella pertussis Infection in mice
Author List:
Nguyen, Alana; Graduate, Biology, San José State University, Presenting Author
Abramson, Tzvia; biology, San José State University
Abstract: Bordetella pertussis (B. pertussis), the causative agent of whooping cough, is a highly contagious respiratory tract disease marked by extensive damage to the mucosal lining and a delay in immune response due to the pertussis toxin (PTX). We focus our study on the characteristics and mechanisms of function of two innate immune cell populations, the dendritic cells (DCs) and the innate lymphoid cells (ILCs). Production of interleukin-22 (IL-22) by type 3 innate lymphoid cells (ILC3) are stimulated by interleukin-23 (IL-23) secreting dendritic cells in response to pathogens and commensals, aiding in the regeneration of the damaged mucosal epithelial barrier and the secretion of antimicrobial peptides (AMPs) to resolve infections. In this study, we hypothesize that infection with B. pertussis interferes with the IL-23/IL-22 axis pathway due to PTX production. To verify our hypothesis, we compared the related immune responses in two mice models of whooping cough, one triggered by B. pertussis and the other triggered by Bordetella parapertussis (B. parapertussis) – a strain that lacks the key virulence factor, the PTX, and causes an acute and fast resolving respiratory disease. Our initial studies using a multicolor flow cytometry analysis show an increased frequency of lung CD103+ conventional DC (cDC), plasmacytoid DC (pDC), and monocyte-derived DC (moDC) detected at 2dpi with B. parapertussis compared to B. pertussis, suggesting a dependent PTX-inhibition of DC recruitment to the lungs. No differential frequency between infections was observed in CD11bhighCD103int alveolar macrophage, suggesting that it is a residential population of the lungs independent of PTX effects. We will next verify if any of the DC populations are compromised in IL-23 secretion. On the other hand, increased but similar levels of ILC3 and ILC2 were observed at 2dpi with both B. pertussis and B. parapertussis-infected C57BL/6 mice compared to uninfected mice, suggesting they are a key population in the innate immune response and perhaps local proliferation of ILCs occurs in the lungs or that recruitment is not PTX dependent. We will next verify if the expression of IL-23 receptor and IL-22 secretion by ILC3 are reduced during infection with B. pertussis. The outcomes of this study may clarify the inability of these innate immune responses to resolve B. pertussis infection early.
Poster #: 232
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Sinorhizobium meliloti, Genetics, Mutant characterization
Project Title: Analyzing the phenotypic consequences of knocking out the sma5027 gene in Sinorhizobium meliloti
Author List:
Dillague, Criselda; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Chen, Esther; Biological Science, California State University, Fullerton
Abstract: Sinorhizobium meliloti is a bacterium that has the ability to form an endosymbiosis with its host plant Medicago sativa. In this endosymbiosis, S. meliloti fixes nitrogen for the plant while obtaining carbon sources in return. To form this relationship, the bacterium employs the ExoS/ChvI two-component signaling pathway. ExoS/ChvI controls the transcription of many genes that influence endosymbiosis formation as well as free-living functions apart from the plant.We investigated sma5027, one of the genes known to be transcriptionally regulated by ExoS/ChvI. The sma5027 gene encodes a putative protein with an unknown function that has orthologs only in a few other alpha-proteobacteria. To investigate the function of sma5027, a knockout strain was generated and its phenotypes were examined. We first examined phenotypes of the free-living sma5027 knockout mutant. Phenotypes were examined by spotting dilutions of bacterial strains onto different types of media to test nutrient utilization, production of the exopolysaccharide succinoglycan, motility, cell envelope integrity, and growth in acidic conditions. Next, we tested whether or not sma5027 is required for endosymbiosis. The symbiosis experiment was performed by inoculating lab-grown host plants with either mutant or wild-type bacteria and assessing symbiosis by measuring nodule formation and plant growth. We found that although knocking out the sma5027 gene results in a slight increase in succinoglycan production in free-living bacteria, plants inoculated with the sma5027 knockout mutant grew just as well as plants inoculated with wild-type bacteria. Thus, the sma5027 gene appears to be important for free-living phenotypes of S. meliloti but is not required for a successful symbiosis. This work was supported by a Maximizing Access to Research Careers grant to CSUF from the National Institutes of Health [5T34GM008612-20] and by NSF grant IOS-0818981 to E.J.C.
Poster #: 233
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Neural crest, cadherin, proliferation
Project Title: The role of Cadherin-11 in motility and proliferation of neural crest cells
Author List:
Camacho, Alberto; Undergraduate, Biology, California State University, Northridge, Presenting Author
Manohar, Subrajaa; Undergraduate, Biology, California State University, Northridge, Presenting Author
Abstract: The neural crest (NC) is a population of stem-like cells that is responsible for forming craniofacial bone and cartilage as well as pigment cells and the peripheral nervous system in all vertebrates. These cells begin as epithelial cells in the neural tube, but detach and migrate throughout the body using a process called the epithelial to mesenchymal transition (EMT). Defects in NC development can lead to common birth defects such as cleft palate and albinism. Previous studies have shown that NC cells express a variety of cell adhesion molecules that control their specification, departure from the neural tube, and migratory ability. Cadherin proteins, which are calcium-dependent cell-cell adhesion molecules, have a well-established role in early embryonic development, differentiation and cell migration. Cadherin-11 (Cad11), a type II cadherin protein has been identified as a migratory cadherin that is crucial for normal NC cell migration and has been linked to the migration of cancer cells during tumor metastasis. Cad11 has also been established as a tumor suppressor because it down regulates cell proliferation to allow for cell migration. Until recently, little has been known about the role of Cad11 in regulating proliferation of migratory NC cells. Our objective with this project is to identify if Cad11 is controlling cell proliferation in migratory NC cells in amniote embryos. To answer this question, we first used immunohistochemistry to identify the spatiotemporal localization of Cad11 in relation to proliferative cells in multiple stages of development and determined that the proliferative cells appear to be lacking Cad11. Next, we performed loss of function experiments by microinjecting a translation blocking morpholino to Cad11, and subsequent immunohistochemistry in chick embryos showed delayed NC specification and an inhibition of NC EMT in Cad11-deficient cells. Our hypothesis is that in the absence of Cad11, EMT is inhibited by increased proliferation of NC cells in the dorsal neural tube, and we intend to test this hypothesis by perturbing Cad11 and performing cell proliferation assays and in vitro migration assays using NC explants.
Poster #: 234
Campus: CSU Long Beach
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Cerebellum, Sexual differentiation, Bdnf
Project Title: Age-dependent Change in Expression of Brain-derived Neurotrophic Factor (Bdnf) in the Developing Mouse Cerebellum
Author List:
Rosales, Yesenia; Undergraduate, Department of Biological Sciences, California State University, Long Beach, Presenting Author
Mota, Thomas; Graduate, Department of Biological Sciences, California State University, Long Beach
Tsai, Houng-Wei; Department of Biological Sciences, California State University, Long Beach
Abstract: A variety of neural functions, such as motor coordination, cognition, and emotion, are highly regulated by the cerebellum, and many of those are sexually dimorphic. However, the molecular mechanism underlying sex differences in cerebellar structures and function remains unclear. Brain-derived neurotrophic factor (Bdnf) is highly expressed in the developing cerebellum and is known to promote dendrite branching in cerebellar Purkinje cells, the largest neuron in the vertebrate central nervous system. Mounting evidence supports the involvement of Bdnf in a variety of neurological disorders and mental illnesses. For example, higher expression of Bdnf mRNA and protein is found in the fetal brain compared to the control in a mouse model of autism, which is four times more likely to occur in boys than in girls. Since a perinatal rise in testosterone secreted by developing testes is responsible for masculinizing brain structures and behaviors as well as considered as a risk factor for autism, we hypothesized that Bdnf expression in the mouse cerebellum during early development might be similarly regulated to be sexually dimorphic. To test our hypothesis, the cerebellum was collected from male and female mice on the day of birth (PN0) and 7 (PN7), 14 (PN14), and 21 days (PN21) after birth (n=8 per sex per age). Total RNA was extracted from each cerebellar sample, followed by measurements of Bdnf and ribosomal protein L13A (Rpl13a) mRNA levels with quantitative RT-PCR. Relative expression of Bdnf was calculated by normalizing Bdnf levels against those of Rpl13a and expressed as fold change versus neonatal females. Our data show that Bdnf mRNA levels significantly decrease on PN7, and then drastically increase on PN21 (p<0.001). In addition, we observed no significant difference in Bdnf expression between the sexes (p=0.312), but a trend of male-biased expression at PN0 (F, 2.00 ± 0.14 vs. M, 2.20 ± 0.16) and female-biased expression at PN14 (F, 2.14 ± 0.14 vs. M, 1.98 ± 0.13). We are currently investigating the potential role of perinatal testosterone and steroid receptors in the regulation of Bdnf expression in the developing mouse cerebellum. In summary, our findings suggest that age- and possibly sex-dependent changes in Bdnf expression might be the mechanism underlying the development of cerebral structure and function.
Poster #: 235
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: antimicrobial peptides, periplasmic protein, overexpression
Project Title: Cloning and overexpression of SapA protein from various gram-negative pathogens
Author List:
Dougherty, Maureen; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Ackroyd, Bryony; University of York, United Kingdom
Wilkinson, Anthony; University of York, United Kingdom
Abstract: SapA (for sensitive to antimicrobial peptides) is a periplasmic peptide binding protein that is part of the SapABCDF transporter system. This system is hypothesized to play a key role in the immunosuppressive effects of virulent Gram-negative bacteria by sequestering and transporting antimicrobial peptides (CAMPs) into the cytoplasm to be degraded. A detailed understanding of SapA, bound and unbound to CAMPs, will be instrumental in the development of novel bactericidal compounds based on interference with Sap transport and peptide degradation. Since characterization of SapA was complicated by its insolubility when overexpressed, the goal of this work was to clone and overexpress SapA homologs from diverse Gram-negative pathogens and test their solubility. The SapA homologs from E. coli, S. Typhimurium LT2, and H. influenzae were cloned using pETFPP_30 as vector. The recombinant clones were introduced into E. coli BL21(DE3), which includes a copy of the T7 RNA polymerase gene under the control of an Isopropyl β-D-1-thiogalactopyranoside (IPTG) regulated promoter. The strains generated were used to test subcellular localization and solubility of the SapA analogs grown and expressed at different conditions. The results showed that in all cases the overexpressed protein was insoluble, possibly as part of inclusion bodies, and remained in the cytoplasmic compartment. Furthermore, subcloning the genes as fusions containing PelB, an heterologous periplasmic secretion sequence, did not modify the results. We conclude that the SapA amino acid sequence is likely responsible for the high tendency to remain insoluble when overexpressed, possibly preventing export to the periplasmic space. Future experiments deleting regions of the SapA sequence may permit us to generate derivatives that remain soluble at the high concentrations achieved in the overexpression assays.
M.D. was funded in part by LA Basin Minority Health and Health Disparities International Research Training Program (MHIRT) 5T37MD001368.
Poster #: 236
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Breast Cancer, PEAK1, Cancer Associated Fibroblasts
Project Title: A novel role for PEAK1 kinase in the tumor-promoting effects of breast cancer associated fibroblasts
Author List:
Hamalian, Sarkis; Graduate, Biology, California State University, Northridge, Presenting Author
Shisgal, Preston; Graduate, Biology, California State University, Northridge, Presenting Author
Agajanian, Megan; Graduate, Biology, California State University, Northridge
Kelber, Jonathan; Biology, California State University, Northridge
Abstract: Breast cancer (BC) is a heterogeneous disease consisting of three subtypes: ER/PR+, HER2+ and TNBC. BC notwithstanding, cancer cells within solid tumors interact with their surrounding microenvironment – a behavior linked to tumor progression. The tumor microenvironment is composed of multiple cell types including, but not limited to, mesenchymal stem cells, immune cells, adipocytes, endothelial cells, and cancer associated fibroblasts (CAFs) as well as the tumor cells themselves. Among the many tumorigenic factors upregulated in the BC tumor microenvironment, we found that PEAK1 (Pseudopodium-Enriched Atypical Kinase 1) is expressed at 3-fold higher levels in the tumor versus normal stroma and at 2-fold higher levels in the tumor stroma of patients whose cancer relapsed. Our group has previously reported on the pro-tumorigenic effects of PEAK1 in breast, pancreatic and colon cancer. Thus, we hypothesize that PEAK1 upregulation in BC stromal cell types promotes tumor progression. Among possible stromal cell types, we show that fibroblast-like cells express the highest levels of PEAK1. These data together with a recent report identifying Pragmin (SGK223), the only known family member of PEAK1, to be upregulated in certain subsets of breast CAFs suggest a role for PEAK1 in tumor-promoting CAF functions. In this regard, we show that PEAK1 overexpression in fibroblast-like mesenchymal stem cells increased HER2+ and ER/PR+ BC cell proliferation/survival. Notably, high PEAK1 expression was identified by Western blotting and qPCR in patient CAFs isolated from all BC subtypes – the ER/PR+ CAFs displayed the highest and lowest PEAK1 expression. Future experiments will employ gene silencing and pathway inhibitors to identify novel intracellular, paracrine and juxtacrine mechanisms by which PEAK1hi CAFs promote tumorigenic behavior in BC cells. This work has the potential to develop novel anti-cancer therapies against both the stromal and tumor cell compartments.
Poster #: 237
Campus: San Francisco State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: microbiology, gene regulation, synthetic biology
Project Title: Efficient regulation of gene expression via synthetic bacterial promoters
Author List:
Aviles, Milo; Undergraduate, Biology, San Francisco State University, Presenting Author
Malinowski, Adam; Graduate, Biology, San Francisco State University, Presenting Author
Ruegg, Thomas; Joint BioEnergy Institute
Thelen, Michael; Joint BioEnergy Institute
Hillson, Nathan; Joint BioEnergy Institute
Chen, Joseph; Biology, San Francisco State University
Abstract: The alpha-proteobacteria Caulobacter crescentus and Sinorhizobium meliloti are both model organisms, used to study cell cycle progression and microbe-host interactions, respectively. These species’ success in distinct ecological niches beset with environmental uncertainties attests to their highly adaptable regulatory systems. In this study, we assessed how effectively eight synthetic promoters could modulate gene expression in these two bacteria, in comparison to two previously characterized promoters that are induced by taurine or anhydrotetracycline. All ten promoters were transcriptionally fused to uidA, which encodes beta-glucoronidase, a reporter enzyme. C. crescentus and S. meliloti strains carrying these transcriptional fusions were grown in the presence and absence of appropriate inducers. Expression levels were then determined by measuring the enzymatic activity of the reporter. Our results show that all eight synthetic promoters can be regulated with a relatively inexpensive inducer. Some of the promoters exhibited particularly large dynamic ranges of expression, with low levels of activity in the absence of the inducer and high levels of activity in its presence. The functionality of select synthetic promoters appears to surpass that of the taurine- or anhydrotetracycline-dependent promoter. These results are consistent with those obtained using red fluorescent protein as a reporter. In addition, we found that sequences between the promoter and the start codon of the reporter gene can significantly influence expression. Our study has clear implications in the engineering of bacterial strains for biotechnology purposes. These synthetic promoters may facilitate tightly regulated gene expression at low cost in industrial settings.
This work was supported by the Arnold and Mabel Beckman Foundation, NIH, DOE, and CSUPERB.
Poster #: 238
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Nervous system, synapse, development
Project Title: Elucidation of the molecular mechanisms that underlie neural circuit formation
Author List:
Watters, Katherine; Graduate, Biological Sciences, San José State University
Varshney, Aruna; Staff, Biological Sciences, San José State University
Shankar, Raakhee; Staff, Biological Sciences, San José State University
Magallanes, Khristina; Undergraduate, Biological Sciences, San José State University, Presenting Author
Madrigal, Angela; Graduate, Biological Sciences, San José State University, Presenting Author
Pollock, Sierra; Undergraduate, Biological Sciences, San José State University
VanHoven, Miri; Biological Sciences, San José State University
Abstract: Our nervous system is a network of neuronal circuits that mediate perception, behavior and thought. To form circuits, neurons must identify their correct synaptic partners among the many neurites in a target region, a process called synaptic partner recognition (SPR). The recruitment of synaptic components must be linked to SPR to ensure the fidelity of neural circuit formation. However little is known about how these processes are linked. Elucidating these molecular mechanisms may aid in understanding neurological disorders, such as autism. To study neural circuit formation, we utilize a trans-synaptic marker called Neuroligin 1-mediated GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP), which fluorescently labels specific synapses in live animals. Using this marker, our group previously found that the UNC-6/Netrin ligand and its receptor, UNC-40/Deleted in Colorectal Cancer (DCC), regulate SPR between PHB sensory neurons and AVA interneurons in C. elegans. Our current research focuses on identifying molecules that act downstream of these recognition molecules, to target synaptic components. We discovered that in animals with mutations in unc-69, the homolog of the human Small Coiled-Coil protein (SCOCO), and unc-76, the homolog of the human FEZ1, synapses between PHB and AVA neurons are severely reduced, indicating a requirement for proper synapse formation. We used two classical genetic techniques to determine if unc-69/SCOCO acts in the unc-40/DCC synaptic pathway. By generating and analyzing the synaptic phenotypes of trans-heterozygotes and double mutants between unc-69/SCOCO and unc-40/DCC, we discovered that unc-69/SCOCO does act in the unc-40/DCC synaptic pathway. Moreover, by generating and analyzing the synaptic phenotype of unc-69/SCOCO animals over-expressing unc-6/Netrin, we found that UNC-69/SCOCO likely functions downstream of UNC-6/Netrin. unc-69/SCOCO and unc-76/FEZ1 are thought to act as kinesin cargo adaptors for synaptic components, and our results indicate the first link in our system between synaptic recognition and targeting of synaptic components. We are currently conducting experiments aimed at understanding which synaptic components are transported by unc-69/SCOCO and unc-76/FEZ1. Our ultimate goal is to elucidate the entire pathway from recognition of synaptic partners to synaptic assembly and maintenance. This work is funded by an NSF RUI to MV (1355202) and an LSAMP fellowship to AM (HRD-0802628).
Poster #: 239
Campus: CSU Fresno
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Filopodia , P. Pastoris, Myosin 10
Project Title: Development of Pichia pastoris as a model system for Myosin X-induced filopodia formation
Author List:
Guaman Tipan, Pablo; Undergraduate, Biology, California State University, Fresno, Presenting Author
Gousset, Karine ; Biology, California State University, Fresno
Abstract: Myosin X (Myo10) is an unconventional actin-based motor protein expressed in several vertebrate tissues. Myo10 has key functions in the formation of filopodia and invadopodia used for cell to cell communication and migration in mammalian cells. Recently, we have demonstrated that Myo10 also plays a critical role in the formation and function of tunneling nanotubes (TNTs), a new form of long distance intercellular communication, important in disease spread. However, little is known about the mechanisms of Myo10 regulation and induction of filopodia. Since mammalian cells already have all of the proteins required for filopodia formation, it is difficult to determine what the minimum protein requirements are in filopodia formation. For instance, Myo10 is critical in the cell cycle and cannot be knock-down in cells without cell death. Thus, in order to better characterize the minimum protein requirements for Myo10- induced filopodia formation, we propose to set up a new model system using the yeast P. pastoris. This organism is well known and easy to manipulate genetically. Furthermore, the lack of Myo10 and naturally occurring filopodia in yeast, despite the presence of a functional actin cytoskeleton will allow us to test if Myo10 by itself is enough to induce filopodia in yeast cells and/or if other proteins are required for these cell protrusions to form.
Here we used a pPICZ vector to insert human Myo10 under the control of the AOX1 promoter. X-33 cells, a wild-type P. pastoris strain, were then transfected with the vector by electroporation and cultivated. Using SDS-PAGE gel electrophoresis, we successfully observed Myo10 expression in the transfected colonies. Further confirmation of the Myo10 expression and its location in yeast cells will be determined by inserting GFP-Myo10 in P. pastoris and observing its expression by fluorescence microscopy. Meanwhile, preliminary data by microscopy using phalloidin-rhodamine staining shows differences in the organization of the actin cytoskeleton in cells expressing Myo10 compared to wild-type P. pastoris. Since no cellular protrusions have been observed, we are currently preparing protoplasts (X-33 cells with their cell wall removed) in order to determine if cell protrusions might be induced by Myo10 expression. Taken together these results indicate that Myo10 can be successfully expressed into P. Pastoris and it, by itself, affects the rearrangement of the actin cytoskeleton present in yeast cells.
Poster #: 240
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Acinetibacter, Klebsiella, Transformation Natural
Project Title: Evidence of interspecies DNA acquisition by a naturally competent Acinetobacter baumannii strain
Author List:
Place, Kori; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Traglia, German; IMPaM (UBA-CONICET)
Melano, Roberto; Public Health Ontario Laboratories, Toronto, Ontario, Canada
Ramirez, Maria Soledad; Biological Science, California State University, Fullerton
Abstract: Over the last few years, the number of antibiotic resistant bacteria prevalent in clinical settings has risen – alarming both scientists and government agencies. Among these dangerous pathogens, the “ESKAPE” group of bacteria is of profound concern, as these microorganisms cause the majority of hospital infections and effectively “escape” available antimicrobial treatments. In this group, “A” represents Acinetobacter baumannii, a pathogen that is associated with severe multi-drug-resistant (MDR) infections with high mortality levels. Hypothesizing that natural transformation, plays a key role in the acquisition of resistance determinant by A. baumannii, we have transformed the carbapenem-susceptible A118 strain with gDNA of a carbepenem-resistance Klebsiella pneumoniae (CRKp) strain (Kb18). Transformation events were confirmed by antimicrobial susceptibility testing. Minimal inhibitory concentrations (MICs) to imipenem (IPM), meropenem (MEM), cefotaxime, ceftazidime, amikacin, gentamicin, kanamycin and tobramycin were determined by the gradient diffusion method (E-test method). Moreover, disk diffusion tests was used to determine the resistance profile of A118 and A118::Kb18 to fifteen antibiotics. The complete genome of the transformant cell was sequenced with Illumina MiSeq-I and Nextera XT DNA library was used for sample preparation. Genomic studies were carried out to expose the acquisition of foreign genomic DNA using different bioinformatics tools including: BLAST (version 2.0), ACT (Artemis), ARG-ANNOT, ISfinder, PHAST (PHAge Search Tool), PlasmidFinder, plasmidSPAdes, variant calling, etc.
Our results showed an increase in the resistance level to all the β-lactam antibiotics tested in A118::Kb18. Surprisingly, the MIC to MEM and IPM increase considerably (6 ug/ml and 8 ug/ml, respectively). Moreover, genomic comparison and genomic analysis of the transformant cell revealed that mobile elements (tnsA, tnsB, IS4, Tn3, intI2, etc) as well as resistance genes such as blaTEM-1, blaOXA73, strA, and strB were acquired.
Many evidences of DNA-uptake by A118 were obtained from the genomic analysis that has also an impact the resistance profile. Our results reinforce the idea that natural transformation could be involved in the increasing emergence of AMR in the threatening pathogen, A. baumannii.
Poster #: 241
Campus: CSU San Bernardino
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Influenza, siRNA, CRISPR
Project Title: Using RNA Technologies to Evaluate Host Factors Important in Influenza Replication and Identify New Therapeutic Targets
Author List:
Thompson, Michael; Graduate, Biology, California State University, San Bernardino, Presenting Author
Newcomb, Laura; Biology, California State University, San Bernardino
Abstract: Influenza virus is the causative agent of flu with significant health and economic impacts worldwide. The virus is controlled with annual vaccine, which has variable efficacy, and antivirals, which become ineffective with use due to selection of resistance. Host proteins are integral for viral replication; a novel approach to treat viral infection centers on targeting host proteins that play an essential role in viral replication but a redundant role for the host cell. RNA technologies can be employed to knockdown or knockout host proteins and establish if they are possible targets for development of novel antiviral therapies. Here we transfected cells with siRNA SMARTpools (Dharmacon) or no siRNA control for 48 hours prior to infection with influenza (Udorn H3N2) for 4 hours. RNA was isolated and analyzed by RT-qPCR to assess relative abundance of target mRNA and influenza RNAs. Influenza RNA expression occurs in the nucleus, so we examined host RNA nuclear export factors, including Nxf1, responsible for mRNA nuclear export, Crm1, responsible for rRNA nuclear export, XpoT, responsible for tRNA nuclear export, and Xpo5, responsible for microRNA nuclear export. Each demonstrated a decrease in mRNA when targeted by siRNA, as expected. We found Nxf1 inhibition results in a severe decrease in viral RNA expression, verifying our previous results. Interestingly, Xpo5 inhibition showed an increase in viral RNA expression, supporting the idea microRNAs function to counter influenza replication. We also targeted two host RNA helicases, UAP56 and URH49, paralogs implicated in influenza replication. Unfortunately, we could not decipher between these two as we observed both UAP56 and URH49 mRNA decreased with either siRNA, revealing the siRNA SMARTpools cross-react. Therefore, we aim to create knockout cell lines using CRISPR-encoding plasmids tar-geting each gene so independent roles can be elucidated. Horizon Discovery has provided CRISPR plasmids encoding five different guide RNAs targeting each gene. Sequence analysis of the guide RNAs show little potential to cross-react and no instances of overlap. While initial experiments did not demonstrate CRISPR activity, we will sort cells transfected with CRISPR plasmid (GFP) to establish if any of the guide RNAs results in DNA alteration. Future studies will continue to use RNA technologies with the aim to identify host factors that can serve as viable antiviral targets to counter influenza.
Poster #: 242
Campus: San Diego State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Nuclear Pore Complex, Molecular Crowding, Transport Proteins
Project Title: The effect of macromolecular crowding on the nucleocytoplasmic molecular transport
Author List:
Torres-Robles, Sara; Undergraduate, Chemistry & Biochemistry, San Diego State University, Presenting Author
Kumeta, Masahiro; Laboratory of Plasma Membrane and Nuclear Signaling, Graduate School of Biostudies, Kyoto Universi
Abstract: The nuclear pore complex (NPC) is a protein complex that is located within the double membrane of the nuclear envelope. It modulates macromolecular transport between the cytoplasm and nucleoplasm, which plays a fundamental role in nuclear and cellular processes such as gene regulation and cell cycle control. Due to the NPC’s crowded environment, there is a minimal amount of free water and unoccupied space, which may influence the macromolecules rate of transport through the pore. We conducted nuclear transport assays to observe the retention of green fluorescent protein (GFP)-tagged importin-beta within the nucleus of digitonin treated HeLa cells. In order to take macromolecular crowding into account in vitro, we used crowding agents, such as bovine serum albumin (BSA) and ficoll, to mimic the intracellular environment. Through these experiments, we observed the influx rate (kin) of GFP-tagged importin-beta increased as the concentration of the crowding agent was increased. This may have resulted from the lack of water (solvent) available in the NPC, which influences the passage of transport proteins by conformational change. Furthermore, the crowded environment in the pore can play a role in the nucleocytoplasmic passage of transport proteins.
Poster #: 243
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Peroxisome Proliferator-Activated Receptors, Lipoprotein Lipase, THP-1 macrophages
Project Title: Lipoprotein lipase and Peroxisome proliferator activated receptors (PPARs) mutually regulate each other in THP-1 Macrophages
Author List:
Christian, Bryan; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Madhwani, Kimberly Rose; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Medh, Jheem; Chemistry and Biochemistry, California State University, Northridge
Abstract: Background: Peroxisome proliferator activated receptors (PPAR) are members of the nuclear-receptor gene super family of transcription factors. Upon activation, PPARs bind to specific DNA sequences called peroxisome proliferator regulatory elements (PPRE) and regulate the transcription of various genes involved in various physiological functions. PPAR-g promotes macrophage lipid accumulation and foam cell formation. This function of PPAR may be facilitated by macrophage lipoprotein lipase, a secreted enzyme that hydrolyzes lipoprotein triglycerides and is believed to be atherogenic. LPL has a PPRE in its promoter region suggesting a role for PPAR in regulating its transcription. Various fatty acid products of LPL’s hydrolytic activity may serve as ligands for PPARs. Thus, we hypothesized that LPL and PPAR may mutually regulate each other in macrophages.
Objective: Our objective was to examine the relationship between macrophage LPL and PPAR-g in THP-1 cells. We examined if down-regulation of LPL can modulate the expression of PPARs and conversely, if modulation of PPAR expression can alter LPL transcription.
Methods: Short hairpin RNA was used to specifically silence either the LPL or PPAR-g gene, and lentivirus particles were used to over-express PPAR-g in THP-1 macrophages. The effect of such gene modulation on the transcription of both LPL and PPAR-g was determined by RT-PCR analysis. Additionally, lipid and protein extracts from WT and LPL-knock-down macrophages were investigated for their ability to drive the activation of luciferase in cells transfected with PPAR-luciferase reporter gene vectors.
Results: RT-PCR analysis showed that the level of PPAR-g transcripts in LPLKD cells was 93% of the level in WT cells. Cellular lipid extracts (a source of agonist/antagonist) from LPL-KD lipids exhibited more PPAR activation than WT lipids. Specific repression of PPAR-g1 resulted in down-regulation of LPL to only 27% of the level in WT cells while over-expression of PPAR-g1 induced the transcription of LPL up to 2 fold in the absence of ligand and up to almost 4-fold when activated with Ciglitazone.
Conclusions: PPAR-g expression levels are directly correlated with LPL transcript levels. Also, it appears that while LPL may not influence PPAR-g expression, lipolysis products of LPL may repress the transcription of PPAR-g-responsive genes.
Acknowledgements: This work was supported by National Institutes of Health Awards R15HL083946 and SC3GM095413.
Poster #: 244
Campus: CSU Fresno
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Phlox, population genetics, polyploidy
Project Title: Does subspecific variation correspond to cytotypic variation in the widespread taxon Phlox speciosa (Polemoniaceae)?
Author List:
Aguilar-Gutierrez, Estefania; Undergraduate, Biology, California State University, Fresno, Presenting Author
Waselkov, Katherine; Biology, California State University, Fresno
Abstract: Phlox speciosa, or showy phlox, ranges from the Sierra Nevada of California into the Coast and Cascade Ranges of the Pacific Northwest (to British Columbia), and into the Rocky Mountains in Idaho and western Montana. It grows at low to middle elevations (100-2400 m), in rocky, wooded slopes and sagebrush habitat, and is easily distinguishable from congeneric taxa that grow in the same habitats by its upright habit, showy flowers, and short length of the style relative to the stigmas. Several subspecies and varieties were identified by previous taxonomists (originally Edgar Wherry in 1955), based on the obvious morphological variation in the group, but this variation does not correspond well to geography, and the current Flora of North America taxonomic treatment has suspended the recognition of subspecific taxa in P. speciosa pending extensive genetic and cytotypic investigation. We are exploring the genetic diversity and connectivity of 20 populations from across the range of this species. Our hypothesis was that P. speciosa would have ploidy level variation between populations, based on its phylogenetic position in Phlox, its broad geographic range, and the huge amount of morphological and ecological variation the species encompasses. The project used flow cytometry, and field sampling of leaf tissue (20 plants per population) was guided by our study of previously collected herbarium specimens. Thus far, our results show no cytotypic variation in the species: all 13 populations sampled to date proved to be diploid (2n=14), as measured by carefully calibrated flow cytometry. Thus, we can conclude that the phenotypic and habitat variability that P. speciosa exhibits are not due to ploidy-level differences leading to intraspecific reproductive isolation. While this finding does not support our hypothesis, it will provide the basis for further exploration of genetic connectivity between these diploid populations using codominant genotyping data from 7 microsatellite markers (previously designed and implemented in other western North American Phlox species). We will continue the project with 7 additional population samples and a new alternative hypothesis: that the observed patterns of morphological and ecological differentiation between populations are due to genetic discontinuity rather than simply phenotypic plasticity. This research will inform important evolutionary questions about species limits and subspecific variation in the genus Phlox.
Poster #: 245
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: gene therapy, neurodegenerative disease, ion channel
Project Title: PAX5 adenovirus-induced expression of Mucolipin-2 in human glial and kidney cell lines
Author List:
Rosas, Lauren; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Cuajungco, Math; Biological Science, California State University, Fullerton
Abstract: The neurodegenerative disease Mucolipidosis type IV (MLIV) is caused by mutations or deletions in the Mucolipin-1 (MCOLN1) gene, which encodes the Transient Receptor potential Mucolipin-1 (TRPML1) protein. MLIV is manifested by motor problems, cognitive dysfunction, low gastric acid production, cataract, and retinal cell death leading to blindness. MLIV has no known cure. The Mucolipin ion channels consist TRPML1, -2, and -3 proteins, which are encoded by MCOLN1, -2, and -3 genes, respectively. They are known to serve as non-selective cation channels located within endosomes and lysosomes. Based on its high homology with MCOLN1, we hypothesize that the MCOLN2 could substitute for the loss of functional MCOLN1 in MLIV. In order to study the potential therapeutic effects of MCOLN2 in MLIV, we must first show that it is expressed in tissues that are mostly affected by MLIV, which is the brain. Previous reports showed that MCOLN1 is widely expressed in tissues, while MCOLN2 and -3 have tissue-specific expression with little to no detectable transcripts in brain tissue. We thus sought to identify the transcription factor responsible for MCOLN2 expression, and discovered the PAX5 protein. The current challenge is to induce the expression of MCOLN2 in brain cells such as neurons or glial cells, which do not produce endogenous MCOLN2, by heterologously expressing PAX5. To demonstrate this proof-of-principle approach, we used an adenoviral vector over-expression system containing the PAX5 cDNA. We then transduced human neuroglioma (H4) cells with PAX5 adenovirus to forcibly induce the expression of MCOLN2. Further, we used human embryonic kidney 293 (HEK-293) cells as positive control based on a priori observation that HEK-293 cells are amenable to PAX5-mediated targeted induction of MCOLN2 expression. Standard reverse-transcription polymerase chain reaction (RT-PCR) revealed MCOLN2 expression in H4 neuroglioma cells by 72 hours post-infection. Real-time quantitative RT-PCR and Western blot analyses both validated the PAX5-induced Mucolipin-2 gene and protein expression, respectively. Overall, we show for the first time that heterologous expression of PAX5 transcriptionally activates and increase the levels of MCOLN2 in a cell type that do not normally or constitutively express the MCOLN2 gene. We will use a human neuronal cell line to further explore the therapeutic potential of gene complementation to rescue the cellular phenotype in MLIV disease.
Poster #: 246
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: immune receptor, virus, 14-3-3 proteins
Project Title: Identifying the Roles for the Tomato Tm-2^2 Immune Receptor and 14-3-3 Gene Family in Effector-Triggered Immunity against Tobamoviruses
Author List:
Sandoval, Carina; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author, Nagel Award Finalist
Spencer, Jennifer; Undergraduate, Biological Science, California State University, Fullerton
Sobhanian, Soha; Graduate, Biological Scienc, California State University, Fullerton
Sacco, Melanie; Biological Science, California State University, Fullerton
Abstract: Effector-triggered immunity (ETI) is a plant defense mechanism in which pathogen effectors are recognized by plant immune receptors that activate defense responses. ETI often limits pathogen proliferation by promoting localized cell death known as the hypersensitive response (HR). Solanum lycopersicum (tomato) possesses the Tm-2^2 immune receptor that confers resistance against Tobamoviruses by detection of the 30kDa viral movement protein(30K). We found that the amino-terminal coiled-coil (CC) domain of Tm-2^2 interacts with a 14-3-3 protein from Nicotiana benthamiana using co-immunoprecipitation and tandem mass spectrometry (MS/MS). Tomato possesses twelve 14-3-3 isoforms, called TFTs. We cloned ten TFT genes for co-expression studies with Tm-2^2 and 30K in N. benthamiana. Nine of the TFTs enhanced HR activation elicited through Tm-2^2 while TFT6 showed the opposite phenotype and inhibited HR. Since 14-3-3 proteins bind phosphorylated sites in proteins, we hypothesized that highly conserved threonine or serine residues at positions 7, 8 and 13 in the Tm-2^2 CC domain are important for signaling involving TFTs. Potential phosphorylation targets were mutated to aspartic acid(T7D, S8D, S13D) as a phosphomimic, or to alanine (T7A,S8A,S13A) and tested mutants in N. benthamiana with 30K. The wild-type or mutant Tm-2^2 CC domain clones were not sufficient to elicit HR in the presence or absence of 30K. For full-length Tm-2^2 immune receptors, the full-length T7A clone retained function and triggered HR in the presence of the 30K protein, showing no effect of the mutation. Surprisingly, the T7D clone failed to elicit HR in the presence of the 30K effector protein, showing loss of function. This evidence supports that position 7 of Tm-2^2 is a phosphorylation target that negatively regulates the activation of the Tm-2^2 immune receptor in the charged state. Negative regulation of immune receptors is important to prevent inappropriate activation that can lead to cell death in the absence of pathogen effector. We propose a model in which TFT6 binds to the phosphorylated T7 in the CC domain of Tm-2^2 in the absence of 30K, maintaining the immune receptor in an inactive state. Recognition of 30K by Tm-2^2 likely leads dephosphorylation of T7 that releases TFT6, and therefore Tm-2^2 signaling inhibition. Elucidating the signaling pathways that function to eliminate pathogen spread is of interest to understand how we can avoid loss of important food crops due to disease.
Poster #: 247
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: sigma / anti-sigma factor, cyanobacteria,
Project Title: Phenotypic evidence of the regulatory role of a sigma/anti-sigma factor pair in Nostoc punctiforme
Author List:
Amir Ali, Arsalan; Graduate, Biology, California State University, Northridge, Presenting Author
Rahaman, Sheera; Undergraduate, Biology, California State University, Northridge, Presenting Author
Fuentes, Nicole; Graduate, Environmental and Occupational Health, California State University, Northridge
Polin, Jenevieve; Staff, Biology, California State University, Northridge
Summers, Michael; Biology, California State University, Northridge
Abstract: The filamentous cyanobacterium Nostoc punctiforme grows using oxygenic photosynthesis and can differentiate into spore-like akinetes, nitrogen-fixing heterocysts, or motile hormogonia in response to stress. A subset of the genes encoding stress responses are hypothesized to be regulated by the 12 alternative sigma factors, some of which are hypothesized to be sequestered by the seven putative anti-sigma factors until stress induces their release. A screen for interaction of all sigma factors with 7 putative anti-sigma factors using a bacterial 2-hybrid assay and found 23 interactions. Quantification of these interactions by β-galactosidase assays showed 12 significant interactions, and two of the 12 with the highest β-galactosidase activity were then confirmed by GST-pull-down assay and Western blot. To better understand gene regulation in cyanobacteria, we conducted phenotypic studies on one of those two sigma/anti-sigma factor pairs, Npun_R0876 (anti-SigB2) and Npun_R4091 (SigB2). A GFP-reporter strain for Npun_R0876 showed increased expression after 72 hours of high light stress (137 µmol photons m-2 s-1), and a GFP-reporter strain for SigB2 showed increased expression after 24 to 48 hours of exposure to envelope stress (0.4 mM EDTA). Stress survival tests conducted on mutant (Δ0876) and overexpression (OE) strains of Npun_R0876 showed that under normal light, Δ0876 grew better than a wild-type (WT) strain (120% of WT growth). Under high light, WT growth was 15% of normal; OE growth, 8%; and Δ0876 growth, 20%. Under EDTA stress, WT achieved only approximately 70% of normal growth, but Δ0876 fared much more poorly, achieving only less than 50% of normal WT growth. Overexpression of Npun_R0876 appeared to have a protective effect against EDTA stress, since the OE strain achieved 80% of normal WT growth. Neutral lipid staining showed that Δ0876 produces significantly fewer lipid droplets than WT, and Δ4091 produces slightly more than WT. In summary, loss of the anti-SigB2 Npun_R0876, which is hypothesized to make more SigB2 available, results in better growth under normal and high light conditions but poor growth under envelope stress. Understanding regulatory control in response to stress and differentiation in cyanobacteria provides essential elements of a bioengineering toolkit to use with these promising organisms for the production of high-value compounds.
Poster #: 248
Campus: CSU Fresno
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Caenorhabditis briggsae, mitochondria, hybrid
Project Title: C. briggsae hybrid developmental delay is caused by mitochondrial-nuclear mismatch
Author List:
Montelongo, Morgan; Undergraduate, Biology, California State University, Fresno, Presenting Author
Ross, Joseph; Biology, California State University, Fresno
Abstract: Like Caenorhabditis elegans, the microscopic nematode C. briggsae is a model system for understanding how genetics influences development, because it is easy to observe development in these individuals. Some C. briggsae F2 generation hybrids, produced by crossing two wild isolate strains, take longer to reach sexual maturity than their wild-type siblings. This retarded development is referred to as “developmental delay”. Previous research indicates that developmentally delayed F2 hybrids also have reduced mitochondrial function. This finding led me to hypothesize that mitochondrial dysfunction in hybrids elicits developmental delay as a result of genetic interaction between one strain’s mitochondrial genome and the other strain’s nuclear genome. To test this hypothesis, my goal was to determine whether the development rate of one type of C. briggsae hybrid, a cybrid (cytoplasmic-nuclear hybrid, in which the nuclear genome of one strain and the mitochondrial genome of another are artificially combined), is slow compared to the parental strains: AF16 and HK104. To achieve this goal, I acquired micrographs of individual worms throughout development. I then made digital measurements of the lengths of each individual to track development rate. The cybrid strain did differ in development rate compared to the two control strains. Future directions will involve repeating this experiment with replicate cybrids before genotyping the cybrid strains to identify the mitochondrial and nuclear alleles that negatively interact in F2 hybrids to produce developmental delay. My research on this putative genetic conflict seeks to address the broader question of how different versions of genes can negatively interact, when combined in a hybrid individual, to cause biological defects.
Poster #: 249
Campus: Sacramento State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Plant, Arabidopsis, Hormone
Project Title: Transcriptional regulation of the tomato H+-ATPase LHA2 by auxin in the control plant growth and development
Author List:
Courtney, Teo; Undergraduate, Biological Sciences, California State University, Sacramento
Driskill, Justin ; Graduate, Biological Sciences, California State University, Sacramento, Presenting Author
Keating, Deidre; Undergraduate, Biological Sciences, California State University, Sacramento
Rechlin, Leah; Undergraduate, Biological Sciences, California State University, Sacramento
Phillips, Hannah; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Ewing, Nicholas; Biological Sciences, California State University, Sacramento
Abstract: Plasma membrane H+-ATPases use the energy of hydrolysis of ATP to pump protons out of plant cells. The pumps participate in many physiological processes and much of their regulation identified to date occurs at the level of protein phosphorylation. One of the key processes in which H+ pumps have been proposed to be critical is in the control of plant growth by the hormone auxin where they have been shown to be regulated by phosphorylation.
The pumps are encoded by a family of genes with 9 to 12 members in all plants that have been examined thoroughly and each is expressed with cell, tissue and developmental specificity. In order to fully understand the control of plant growth and development by auxin it is important to identify which isoforms are expressed in auxin responsive sites and to define how they are regulated.
We fused the promoter of the tomato isoform LHA2 to the reporter gene GUS and transferred this construct into both Arabidopsis and tomato. We observe that it is expressed in key auxin responsive sites and that its level and pattern of expression change in response to natural and synthetic auxins and auxin transport inhibitors. Further, we observe that the promoter includes 3 auxin response elements (AuxREs) with the sequence TGTCTC that have been shown to confer auxin responsiveness to other genes and synthetic constructs including the well-characterized synthetic DR5:GUS construct that is driven by a tandem array of 7 TGTCTC’s. To test whether these elements confer auxin responsiveness to LHA2 we used site-specific mutagenesis to inactivate these elements by changing a single base that has been demonstrated to be essential for function of the element. These changes reduce, but do not abolish, auxin responsiveness indicating both that these elements participate in the control of LHA2 expression and that there likely are additional auxin response elements present in the promoter. Histochemical detection of expression shows that down-regulation of the mutagenized promoter occurs primarily in the root vasculature in the elongation zone and not in the root apical meristem where DR5:GUS is most highly regulated. This indicates that the AuxREs are necessary but not sufficient for auxin responsiveness in the elongation zone vasculature. We are currently conducting qRT-PCR to test for changes in LHA2 mRNA levels in tomato in response to auxin.
This work is funded by the Albert Delisle Family and CSUS Associated Students, Inc.
Poster #: 250
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila, nicotine, development
Project Title: The effects of larval exposure to nicotine on development in Drosophila melanogaster
Author List:
Wong, Lisa; Graduate, Biological Sciences, San José State University, Presenting Author
Okeke, Ifechukwu; Undergraduate, Biological Sciences, San José State University
French, Rachael; Biological Sciences, San José State University
Abstract: Tobacco exposure during pregnancy is associated with a wide range of birth defects and conditions including delayed development, slow growth, low birth weight, mental retardation, and decreased survival. Nicotine, tar, and carbon monoxide are three of the most well-characterized harmful chemicals in tobacco smoke, and while the effects of these chemicals on development are clear, and the direct target of nicotine is known (nicotinic acetylcholine receptors), the mechanisms underlying the deleterious effects of nicotine on development are nonetheless not well-understood. In addition, many of the developmental effects of nicotine exposure in mammals overlap with those of developmental alcohol exposure, suggesting common downstream target pathways.
We are using the genetic model organism Drosophila melanogaster (the common fruit fly), to model developmental nicotine exposure. Flies are allowed to develop on food containing increasing concentrations of nicotine, leading to exposure throughout larval development. We find that nicotine has a dose-responsive effect on both survival and development time. A concentration (in food) of 0.01% reduces survival to 60% of wildtype levels, and increases development time by approximately 12 hours, while at a concentration of 0.05%, all animals died as first instar larvae. Finally, we see a trend towards reduced body size in animals developing on 0.01% nicotine.
Having established a dose-responsive developmental effect of nicotine, we are now in the process of characterizing the interaction between nicotine and ethanol during development, as well as examining the effects of moderate-dose nicotine exposure on the development of the central nervous system (CNS). We will present the results of these experiments.
Poster #: 251
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Sinorhizobium meliloti, Molecular Biology, Electrophoretic Mobility Shift Assay
Project Title: Testing the DNA binding site recognized by the ChvI response regulator in the nitrogen-fixing bacterium Sinorhizobium meliloti
Author List:
Mendoza Cavazos, N. Carolina; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author, Nagel Award Finalist
Chen, Esther; Biological Science, California State University, Fullerton
Abstract: Nitrogen is the most abundant element in the atmosphere, but the dinitrogen must be reduced, or fixed, before it can be used by plants. Sinorhizobium meliloti bacteria can reduce atmospheric nitrogen to ammonia in a process known as biological nitrogen fixation, and in exchange they receive dicarboxylic acids from their plant host, Medicago sativa. For the symbiosis to occur, both symbiotic partners must orchestrate many changes in gene expression. Bacteria commonly use two-component signaling pathways to control transcription in response to stimuli. The two components are a sensor histidine kinase, which senses a signal and autophosphorylates, and a response regulator, which is phosphorylated by the histidine kinase and consequently regulates transcription. The S. meliloti ExoS/ChvI two-component signaling pathway is required for forming the symbiosis with M. sativa, since it regulates many genes involved in the symbiosis as well as in the free-living form of the bacteria. ExoS/ChvI direct target genes are those genes whose upstream region is bound by the ChvI response regulator to regulate their transcription. Using bioinformatics, our lab previously identified a 14-bp long consensus sequence in the DNA sequences upstream of ExoS/ChvI direct target genes. To determine whether this motif is important for ChvI binding in vitro, we performed Electrophoretic Mobility Shift Assays (EMSA) using purified ChvI protein and the DNA regions upstream of two ExoS/ChvI direct target genes, SMc01580 and ropB1. We found that deletion of the consensus sequence from these upstream regions prevented ChvI binding to the DNA. Furthermore, we found that substitution mutations in the most highly conserved positions in the motif also prevented ChvI binding to the DNA. Our results indicate that the 14-bp long consensus sequence is critical for ChvI binding and that the formation of the ChvI-DNA complex relies on the recognition of highly conserved base pairs within this motif. This work was supported by a Maximizing Access to Research Careers grant to CSUF from the National Institutes of Health [5T34GM008612-20] and by NSF grant IOS-0818981 to E.J.C.
Poster #: 252
Campus: San Diego State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila, skeletal muscle, myopathy
Project Title: The Effects of Protein Expression Manipulation on Muscle Structure and Function in Inclusion Body Myopathy-3
Author List:
Manalo, Kimberley; Graduate, Biology, San Diego State University, Presenting Author
Suggs, Jennifer; Staff; Biology, San Diego State U, Biology, San Diego State University
Melkani, Girish; Graduate, Biology, San Diego State University
Melkani, Anju; Staff, Biology, San Diego State University
Bernstein, Sanford I; Biology, San Diego State University
Abstract: Inclusion body myopathy-3 (IBM-3) is a dominant human disease causing progressive degradation of the muscle. IBM-3 is caused by a missense mutation in myosin heavy chain (MyHC) type IIa that changes glutamic acid at position 706 to a lysine residue.
Our lab has created a transgenic Drosophila model that mimics the human disease by expressing the analogous mutation E701K. Proteomic analysis of the insoluble protein fraction derived from indirect flight muscle (IFM) lysates in homozygous mutant flies were compared to wild type flies at young and old ages to determine differences in protein expression. Six proteins were over-expressed and twelve proteins were under-expressed in the protein aggregates in the mutant flies at both ages. While the functions of several proteins are unknown, others serve structural, metabolic, protein folding, protein turnover, signaling, synthesis, or transcription roles.
I am testing the hypotheses that a) these proteins play a critical role in wild-type muscle development and function and b) that manipulating the expression level of these proteins will lead to improvement or detrimental effects on the phenotype of the E701K/+ mutant flies. To address these hypotheses, I use the UAS-GAL4 system to modulate expression of the identified genes in the wild-type and E701K/+ backgrounds, using flight ability as a readout of IFM function. I will then use fluorescence microscopy to assess whether modulating protein levels lead to structural differences in the IFM.
I used the IFM-specific driver lines (88F-GAL4 and fln-GAL4) in conjunction with RNAi to knock-down each gene in IFM. Flight impairment was observed in young flies with the 88F-GAL4 driver line coupled with knock-down of CG8764 (oxen) or CG15105 (abba/tn). Further, progressive decline was seen by over-expression of CG4463 (Hsp23). I produced three additional over-expression constructs by cloning cDNA of the genes of interest into the pUASTattB plasmid, and will use GAL4 induction to drive their expression. Currently, our data suggest that abba/thin, oxen, and Hsp23 play a role in muscle development in the wild-type fly. Future studies will determine if modulating expression levels of these proteins causes structural changes in the wild-type muscle and if there is an effect in E701K/+ mutants.
Overall, these studies will lead to an understanding of the roles that specific aggregation-prone proteins play in normal and diseased muscle structure and function.
Poster #: 253
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: natural variation, heat-shock proteins, selection
Project Title: Purifying selection explains the evolution and natural variation of HSPA1A, the major stress inducible gene, in humans
Author List:
Ord, Sara; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Nguyen, Peter; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Nikolaidis, Nikolas; Biological Science, California State University, Fullerton
Abstract: HSPA1A is the major stress inducible 70-kDa Heat shock protein (Hsp70) in humans. This protein is a critical component of the cellular stress response and alterations in its function have been associated with several diseases, such as cancer, and neurodegenerative conditions. However, the extent of HSPA1A’s natural variation within and between species remain largely unknown. Here, we investigated how this gene evolved in humans and other mammals. Our results revealed that in all Eutherian mammals HSPA1A is actually two almost identical proteins coded by two tandemly arranged genes. This extreme level of sequence conservation, both at the nucleotide and protein levels, can be explained by a gene conversion-like mechanism that resulted in sequence homogenization. However, gene conversion alone cannot explain the observed evolutionary pattern of all the mammalian heat-inducible genes, which contain several non-amino acid altering (synonymous) mutations. Therefore, we conceptualized that both gene conversion and purifying selection act to conserve these Hsp70 genes. This notion is also supported by analyses of HSPA1A variation in humans, which revealed that all mutations have a very low frequency and that the majority of these mutations is synonymous. The presence of purifying selection was further revealed by determining whether natural variants affect the major chaperone functions of HSPA1A. Specifically, recombinant proteins corresponding to the wild-type (WT) and five mutated variants were tested for their ability to hydrolyze ATP. These experiments revealed that the mutations did not significantly alter the rate of ATP hydrolysis. Then Isothermal Titration Calorimetry was employed to test whether the mutations alter binding of HSPA1A to ATP, ADP, and protein substrates. These experiments showed that only one mutation increased the entropy of the reactions. Lastly, the effects of these mutations on protein stability was determined by calculating the protein’s melting temperature (Tm). This assay revealed that three mutations had significantly higher Tm as compared to the WT protein. These experiments suggest that although purifying selection acts to preserve the primary functions of HspA1A, other secondary functions of the molecule may be altered. Collectively, our results constitute an evolutionary paradigm in which gene conversion, purifying selection, and neofunctionalization act conversely to conserve a critical component of the heat shock response.
Poster #: 254
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Apoptosis, Autophagy, Glucocorticoids
Project Title: Interactions between Apoptosis and Autophagy in Glucocorticoid –evoked death of human leukemic CEM cells
Author List:
Zhou, Shuo; Undergraduate, Biology, California State University, Northridge, Presenting Author
Ortega, Carina; Undergraduate, Biology, California State University, Northridge, Presenting Author
Medh, Rheem; Biology, California State University, Northridge
Abstract: Glucocorticoids (GCs) bind to GC receptors and initiate a pathway for programmed cell death (PCD) in GC-sensitive human leukemic CEM cell lines. While GCs are widely thought to induce Type I PCD (apoptosis), recent studies suggest overlapping influences of Type II PCD (autophagy). Autophagy has a dual role in cell death: it can (a) digest damaged cellular organelles to prevent cell death from apoptosis; (b) enhance cell death along with apoptosis. The synthetic GC dexamethasone (Dex) kills CEMC7-14 cells by apoptosis via upregulation of E4BP4 and Bim, while CEMC1-15 cells are resistant to Dex. Ectopic expression of mouse E4BP4 (CEMC1-5mE#3 cells) sensitizes CEMC1-15 cells to Dex-mediated responses. Studies presented here tested the hypothesis that Dex-mediated activation of autophagy blunted or inhibited apoptosis in CEM cells, and that inhibiton of apoptosis could divert cells toward autophagic death.
All three CEM cell lines were treated with Dex, the apoptosis inhibitor NS3694 (inhibits apoptosome formation and prevents caspase activation), or the autophagy inhibitor Cpd18 (inhibits PI3K pathway to block autophagosome formation), either alone or in combination for evaluation of cell viability by Trypan blue dye exclusion and MTT assays. A hallmark of apoptosis is accumulation of cells in sub-G1 phase of the cell cycle. Flow cytometric analysis of cell cycle was performed to estimate the percentage of apoptotic cells.
The MTT assay indicated that inhibiting autophagy by Cdp18 significantly promoted Dex-induced cell death in the sensitive cells, but did not improve responsiveness of resistant CEMC1-15 cells to Dex. In contrast, the apoptosis inhibitor NS3694 significantly increased cell viability /proliferation in all three cell lines. Flow cytometric cell cycle analysis confirmed that the cell death represented type I PCD (apoptosis).
Results suggest that NS3694 is an effective antiapoptotic agent. Inhibition of autophagy potentiated Dex-mediated apoptosis in the sensitive cells suggesting that autophagy and apoptosis represent mutually antagonistic responses within cells. However, since blocking autophagy did not restore Dex sensitivity in CEMC1-15 cells, resistance to both pathways may be coupled and mediated by a common intermediate(s).
Acknowledgements: This research is funded by a NIH SCORE SC3 award to RDM (GM081099). The authors acknowledge the support of the CSUN College of Science and Mathematics.
Poster #: 255
Campus: CSU Los Angeles
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: CRISPR/Cas9, proteasome, wdr68 dyrk1a
Project Title: Wdr68/Dcaf7 is required to stabilize Dyrk1a protein and function
Author List:
Xu, Jingyi; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Yousefelahiyeh, Mina; Graduate, Biological Sciences, California State University, Los Angeles
Yu, Yang; Graduate, Biological Sciences, California State University, Los Angeles
Alvarado, Estibaliz; Staff, Biological Sciences, California State University, Los Angeles
Nissen, Robert; Biological Sciences, California State University, Los Angeles
Abstract: WD40-repeat domain protein Wdr68/Dcaf7 is essential during embryonic development for craniofacial patterning and functions as a molecular scaffold to organize the activity of multiple kinases. Dual-specificity Tyrosine-regulated Kinase 1A (Dyrk1a) physically interacts with Wdr68, is overexpressed in Down Syndrome patients, and is essential for embryonic development. Dyrk1a can also act as a nuclear transcriptional co-activator by phosphorylating the Carboxyl-Terminal Domain (CTD) of RNApII. Wdr68 might act during development by facilitating the kinase functions of Dyrk1a with impacts on downstream gene expression. Here we found that a Gal4DBD-Wdr68 fusion protein minimally (2-fold) activated a 5xGal4-Luciferase reporter in transiently transfected mouse C2C12 cells maintained in growth medium (GM). In contrast, the Gal4DBD-Wdr68 fusion activated the 5xGal4-Luc reporter 18-fold when cells were maintained in differentiation medium (DM). Western blot analysis revealed strong up-regulation of endogenous Wdr68 and Dyrk1a protein levels in cells maintained in DM versus GM suggesting that the increased levels of Dyrk1a CTD-kinase activity might underlie the mechanism of Gal4DBD-Wdr68 activity. We then generated Wdr68 deletion (Δwdr68) sublines by CRISPR/Cas9 gene targeting in C2C12 cells. We found a near-complete loss of Dyrk1a protein expression in Δwdr68 sublines relative to a non-target (NT1) control subline. We treated the Δwdr68 sublines and NT1 controls with the proteasome inhibitor LLNL. We found that 6 hours of 50uM LLNL treatment could restore near-normal expression levels of full-length Dyrk1a in Δwdr68 sublines. We also generated Dyrk1a deletions by CRISPR/Cas9 gene targeting in C2C12 cells and found near normal levels of Wdr68 expression in them suggesting the stabilization is unidirectional as Wdr68→Dyrk1a. Overexpression of a GFP-Wdr68 fusion protein increased the level of Dyrk1a in both NT1 and ΔWdr68 sublines. Thus, Wdr68 is required for stabilization of Dyrk1 kinases and may represent a novel therapeutic target for the modulation of Dyrk1a activity. To assess how general the Wdr68→Dyrk1a stabilization mechanism might be, we also generated Δwdr68 sublines of the human HeLa cell line. A similar requirement for Wdr68 was found for Dyrk1a in HeLa cells. Thus, Wdr68 stabilizes Dyrk1a in both mouse and human cells and likely represents a general role for Wdr68. This work is supported by grants from CSUPERB and the NIH-NIDCR.
Poster #: 256
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila, evolution, heart
Project Title: Comparison of heart function between short-lived and long-lived populations of Drosophila melanogaster
Author List:
Ferrer, Claudia; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Shahrestani, Parvin; Biological Science, California State University, Fullerton
Abstract: The function of the heart is vulnerable to diseases and dysfunction as seen in humans and also in the model organism, Drosophila melanogaster. Heart function was compared between ten fly populations: five populations with accelerated developmental period, which lived shorter lives, and five control populations, which had longer developmental period and lived longer lives. An electrical pacing assay was used to stimulate the hearts of flies to induce cardiac arrest in the short-lived and long-lived populations, and heart failure rates were recorded after pacing. Microscopic video recording of the flies’ heart beats were used to measure heart period, arrhythmia index, systolic and diastolic intervals and diameters, and fractional shortening. In general, short-lived populations showed significantly more heart dysfunction compared to the longer lived control populations. These results indicate that laboratory selection for accelerated development in D. melanogaster populations leads to increased heart dysfunction. Using whole-genome sequence analyses of the short-lived and long-lived populations, some loci that affect heart function were identified.
Poster #: 257
Campus: Cal Poly San Luis Obispo
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: mRNA decay, phosphatase, co-immunoprecipitation
Project Title: Dephosphorylation of the RNA Binding Protein Tristetraprolin by Protein Phosphatase 2A in Lipopolysaccharide Stimulated Murine Macrophages
Author List:
Wampler, Derek; Graduate, Biological Sciences , California Polytechnic State University, San Luis Obispo, Presenting Author
Piñon, Francisco ; Allan Hancock College , Presenting Author
Clement, Sandra; Biological Sciences, California Polytechnic State University, San Luis Obispo
Abstract: The post-transcriptional regulation of gene expression by post-translational modification of RNA binding proteins controls turnover rates of cytokine transcripts important for inflammation, autoimmune diseases and cancer. Reversible phosphorylation of the RNA binding protein Tristetraprolin (TTP) alters the stability of several of these mRNA transcripts including pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α). While unphosphorylated TTP is able to recruit mRNA decay enzymes to destabilize target transcripts, phosphorylation triggered by the p38/MAPK/MK2 kinase pathway in response to lipopolysaccharide (LPS) exposure leads to mRNA stability in macrophages. The cause and consequences of TTP phosphorylation have been studied extensively, but much less is known about the mechanism and regulation of its dephosphorylation, although the heterotrimeric protein phosphatase 2A (PP2A) has been implicated. In this study, time course experiments were performed to determine the pattern of TTP expression and phosphorylation status in a murine macrophage cell line (RAW 264.7) stimulated with LPS. SDS-PAGE and western blotting analysis demonstrated that TTP expression peaked at 12 hours, and dephosphorylation started around 16 hours after LPS induction. We next examined by western blotting the relative abundance of components of the PP2A enzyme including the catalytic (C) and three candidate regulatory subunits (B, B’ and B’’) of the heterotrimer. We found that PP2A C, B and B’’ protein levels remained relatively constant throughout the time course while the expression of PP2A B’ (B56) was observed only at 12 and 14 hours after LPS stimulation. In order to characterize the interaction between TTP and PP2A at various time points during progression of the cellular response to LPS, we developed an immunoprecipitation assay to purify TTP from LPS stimulated RAW 264.7 cells. Our initial immunoprecipitations indicated successful purification of TTP from LPS-stimulated, but not unstimulated cells. However, PP2A-C co-purified in our assay in the absence of TTP expression as well, which indicated a need for increased stringency in our protocol. Experiments are currently underway to optimize the specificity of this procedure. This work was supported by CIRM Bridges Grant TB1-01175 and the NIH Division of Minority Opportunity in Research Bridges to the Baccalaureate Grant.
Poster #: 258
Campus: San Diego State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: 16S rRNA , polycystic ovary syndrome , high-throughput sequencing
Project Title: Letrozole Treatment of Adult Female Mice Does Not Result in the Full PCOS Metabolic Phenotype or Decreased Gut Microbial Diversity.
Author List:
Torres, Pedro J.; Graduate, Cell and Molecular Biology, San Diego State University, Presenting Author
Skarra, Dana V.; , Department of Reproductive Medicine, UCSD
Anvar, Arya R.; Undergraduate, Department of Reproductive Medicine, UCSD
Thackray, Varykina G., Department of Reproductive Medicine, UCSD
Kelley, Scott T.; Biology, San Diego State University
Abstract: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among women of reproductive age with an estimated prevalence of 5-15%. Women with PCOS have reproductive and metabolic abnormalities that result in an increased risk of infertility, type-2 diabetes and cardiovascular disease. Research has shown that the complex microbial community in the large intestine (gut microbiome) may be a causative factor in the development of metabolic disorders such as obesity and type-2 diabetes. A mouse model was established that recapitulates many of the reproductive and metabolic hallmarks of PCOS by treating pubertal (4 week-old) female mice with the aromatase inhibitor, letrozole. We also recently demonstrated significant, diet-independent changes in the gut microbiome of letrozole-treated mice compared to placebo including a significant decrease in overall species richness and phylogenetic diversity. In this study, we investigated whether treatment of adult female mice with letrozole resulted in similar alterations in reproductive, metabolic and microbial phenotypes compared to pubertal mice. Eight week-old mice were implanted subcutaneously with a placebo or letrozole pellet. Fecal samples were collected and body weight was measured on a weekly basis. A glucose tolerance test was performed on the mice after 5 weeks of placebo or letrozole treatment. We also weighed the parametrial fat pads, measured serum hormone levels and assessed ovarian morphology. Gut microbial diversity profiles were analyzed using culture-independent sequencing of bacterial 16S rRNA gene sequences and QIIME. Our results showed that, similarly to pubertal mice, letrozole treatment of adult female mice resulted in reproductive hallmarks of PCOS including elevated testosterone levels, anovulation and polycystic ovaries. However, in contrast to pubertal mice, treatment of adult mice with letrozole did not result in many of the metabolic hallmarks of PCOS. Furthermore, unlike pubertal mice, treatment of adult mice with letrozole resulted in a significant increase in overall species richness and phylogenetic diversity. In summary, we show that, while elevated testosterone may be required for PCOS, it is not sufficient for developing the full metabolic/microbiome phenotype in adult female mice. Our results also suggest that puberty may be important for development of the PCOS metabolic phenotype in women.
Poster #: 259
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: multidrug resistance, bacterial motility, cell polarity
Project Title: Overexpression of a bacterial efflux pump causing multidrug resistance, reduced motility, and ectopic pole formation in Caulobacter crescentus
Author List:
Arvizu, Ignacio; Undergraduate, Biology, California State University, Northridge, Presenting Author
Wolman, Ram; UCSF Medical School
Murray, Sean; Biology, California State University, Northridge
Abstract: Bacterial fatty acid biosynthesis can be inhibited with the antibiotic cerulenin. In Caulobacter crescentus, obstruction of fatty acid biosynthesis has been linked with many cell cycle regulated events. A previous study linked cerulenin resistance in Pseudomonas aeruginosa with a multidrug efflux pump. To determine if C. crescentus uses a similar mechanism, we isolated spontaneous, cerulenin-resistant, mutants. Genomic sequencing revealed that one mutant had a truncated TetR repressor-like protein called TipR. TipR has been shown to repress the expression of the acrAB2-nodT efflux pump operon. Transcriptional reporters revealed that truncated TipR in our cerulenin-resistant mutant yields increased expression of the efflux pump operon. Additionally, the cerulenin-resistant mutant and an efflux pump overexpression strain were less susceptible to multiple antibiotics (Kirby-Bauer disk diffusion assay), had reduced motility (soft agar assay), and a subpopulation of cells exhibited ectopic pole formation (microscopy). A tipR knockout strain produced similar phenotypes, which can be suppressed by a spontaneous loss-of-function mutation in the acrB2 efflux pump gene. As expected, deleting the efflux pump genes in the cerulenin-resistant mutant restored antibiotic sensitivity, motility, and blocked ectopic pole formation. Ultimately, this confirmed our hypothesis that multidrug resistance, reduced motility, and increased ectopic pole formation resulted from overexpression of the C. crescentus acrAB2-nodT efflux pump.
This work was supported by NIH grant SC2 GM084860 to SM, a CSUN Research, Scholarship and Creativity Award to SM, and NIH R25 GM063787 to MariaElena Zavala in support of IA and a CSUN Presidential Scholarship to RW.
Poster #: 260
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: osteoarthritis, stem cell, cartilage
Project Title: Investigating Prg4 regulation in articular cartilage models using a genetic and stem cell-based approach
Author List:
Daniels, Ryan; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Cutia, Thomas; Boston Children’s Hospital, Harvard Medical School, Boston, MA
Smits, Patrick; Boston Children’s Hospital, Harvard Medical School, Boston, MA
Craft, April; Boston Children’s Hospital, Harvard Medical School, Boston, MA
Abstract: Osteoarthritis (OA) is a degenerative disease that effects the articular cartilage of joints and is a leading cause of disability in the United States. Articular cartilage is identified mainly by the expression of lubricin an extracellular protein. Other groups have shown that lubricin overexpression in the cartilage of transgenic mice is protective against articular degradation following induced join-destabilization. Based on this protective nature, we hypothesize that lubricin could be positively modulated as a potential therapeutic approach.
We took both a genetic and a stem cell-based approach to understand how the gene that encodes lubricin (Prg4) is regulated. We have identified multiple highly conserved regulatory elements within the upstream region of the Prg4 coding sequence, one of which is sufficient to drive Prg4 expression in the joints of developing mice using a non-cartilage related Thymidine kinase (TK) minimal promoter (TK-4X). Seven potential enhancer elements of the Prg4 gene appear to work in synergy with this element in mouse embryonic joints.
In order to generate tools that can be used to identify transcription factors that bind to the regulatory elements, constructs in which combination of these elements and the non-cartilage TK promoter driving eGFP were generated. These constructs were transfected into both mouse embryonic stem cells (mESCs) and an articular chondrocyte-like cell line, MMA2. We used a mESC line generated by collaborators in which a tdTomato reporter has been inserted in the Prg4 locus. We optimized our lab’s published protocol to efficiently differentiate these cells into articular cartilage and confirmed Prg4-tdTomato expression upon articular cartilage differentiation by both microscopy and flow cytometry. We will soon determine whether the reporter constructs behave similarly to the endogenous Prg4 locus by comparing eGFP signal to the tdTomato signal in clonal mESC dual reporter lines. For the MMA2 cells, we found that cells transfected with the TK-4X construct had more intense eGFP signal than cells transfected with a vector in which TK alone drives eGFP expression.
These cell lines can now be used to test candidate transcription factors and signaling molecules that could modulate lubcricin expression. Understanding the mechanisms that regulate lubricin expression in articular cartilage could provide key insights into translation research using novel therapies to treat patients with OA. Funding: HHMI ExROP
Poster #: 261
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: whooping cough, Th7/regs cells, immune response, ,
Project Title: Phenotyping a hybrid Th17-like Treg cell population that arises during whooping chough infection.
Author List:
Gates, Hillary; Undergraduate, Biology, San José State University, Presenting Author
Smith, Andrew A; Graduate, Biology, San José State University
Silva, Elvia; Undergraduate, Biology, San José State University
Bui, Khoa; Undergraduate, Biology, San José State University
Abramson, Tzvia; Biology, San José State University
Abstract: Whooping cough is a highly contagious respiratory disease caused by the bacterium Bordetella pertussis (B. pertussis). Incidence of the disease continues to rise in both the United States and globally, proving especially harmful towards young children and infants. Th17 cells are known to play a central role in the resolution of the infection, yet the bacteria’s toxins attenuate their response and delay their appearance. This could be a reason for the hallmarked prolonged nature of this disease. Launching a proper response to resolve this infection requires orchestration of many immunological cell types. Treg are classically immunosuppressive; however, some research groups have shown the hybrids to be pro-inflammatory, helping resolve infections.
We hypothesize that, during whooping cough infection, Tregs develop a Th17-like effector phenotype in the lungs to help resolve the infection. Using multi color flow cytometry, we have identified a Th17-like hybrid population of cells that arise during late whooping cough infection in mice (~4% of total Th cells). By both in vitro differentiation assays and in vivo knockdown studies, we have confirmed a role for IFNα in the development of this population. Further analyses will interrogate the nature of these cells and evaluate their suppressor versus effector functions ex vivo. By shedding light on the origin of the hybrid population and subsequent cellular relationships between B. pertussis pathogenesis versus resolution during the whooping cough infection, we hope to improve current vaccinations for the deadly disease.
Poster #: 262
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: antisense , RNase P, Locked nucleic acids
Project Title: Optimization of configurations and chemistries of bridged nucleic acids-containing oligomers as external guide sequences
Author List:
Jani, Saumya; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author, Nagel Award Finalist
Jackson, Alexis; Graduate, Biological Science, California State University, Fullerton
Davies-Sala, Carol; Fundacion Instituto Leloir
Zorreguieta, Angeles; Fundacion Instituto Leloir
Tolmasky, Marcelo; Biological Science, California State University, Fullerton
Abstract: Background: External Guide Sequence (EGS) technology has the potential to serve in the treatment of many diseases by inhibiting expression of key proteins. EGSs are antisense oligomers that elicit RNase P-mediated degradation of a target mRNA. Nuclease-resistant analogs must be used to prevent degradation of the EGSs before they exert their action. We tested different configurations of locked nucleic acids (LNA)/DNA and bridge nucleic acids (BNA)/DNA chimeric oligomers to identify the most active. In addition, one of the oligomers was covalently bound to a cell penetrating peptide (CPP) to assess its activity as EGS in growing bacterial cells.
Methods: EGS activity of LNA/DNA and BNA/DNA chimeric oligomers with various configurations that target regions of two different mRNA molecules was determined in in vitro RNase P degradation assays. The reactions included the RNase P components M1 and C5, labeled mRNA, and the appropriate EGS. The products were analyzed by 5% denaturing GTG-PAGE. The action of a CPP-conjugated LNA/DNA chimeric oligomer was tested by addition to bacterial cultures.
Results: All EGSs were 17 residues long and the configurations used were gapmers (LNA-DNA-LNA) – 5L-7D-5L (G1); 5L-8D-4L (G2); 5L-9D-3L (G3) – and one mixmer (3L-6D-3L-2D-3L). Activity of the mixmers that targeted the resistant genes aac(6’)-Ib and cat mRNA was low. Activity of the gapmers differed among the different mRNA molecules. The G2 was more active than G1 and G3 in targeting the aac(6’)-Ib mRNA. G1 was more active than G2 in targeting the cat mRNA, while G3 targeting the cat mRNA demonstrated no cleavage. The CPP-conjugated G2 that targets the amikacin-resistance aac(6’)-Ib mRNA reduced growth of a clinical strain of A. baumannii one added in combination with amikacin.
Conclusions: LNA/DNA gapmers are active as EGSs while mixmers have very low activity. All BNA/DNA chimeric compounds tested showed poor EGS activity. The most active LNA/DNA gapmer configuration seems not to be universal. CPP-conjugated LNA/DNA molecules penetrate bacterial cells and conserve their EGS activity.
Funding: Grants 2R15AI047115-04 and LA Basin Minority Health and Health Disparities International Research Training Program (MHIRT) 5T37MD001368 from the National Institutes of Health.
Poster #: 263
Campus: CSU Long Beach
Poster Category: Product-focused Innovation
Keywords: Alzheimer’s Disease, Therapeutic Drugs, Butyrylcholinesterase
Project Title: Dialkyl Aryl Phosphates: A potential Therapeutic for the Treatment of Alzheimer’s Disease
Author List:
Takenka, Show; Graduate, Chemistry & Biochemistry, California State University, Long Beach, Presenting Author
Acey, Roger; Chemistry & Biochemistry, California State University, Long Beach
Abstract: Alzheimer’s disease (AD) is associated with elevated levels of brain butyrylcholinesterase (BuChE) activity. The result is lower amounts of acetylcholine and lack of cognitive function. In addition, BuChE is associated with neurotoxic beta-amyloid plaques. Thus, BuChE is considered a therapeutic target for the treatment of AD. Tetraethyl-, tetrabutyl-, and tetraphenyl hexyl bisphosphate were tested for their inhibitory activity on BuChE. The compounds are highly selective for BuChE. They do not inhibit acetylcholinesterase or trypsin, enzymes having the same catalytic mechanism as BuChE. BuChE activity was measured by the method of Ellman. Tetraethyl hexyl bisphosphate was the least potent with an IC50 of 10-4 M. Tetrabutyl hexyl bisphosphate has an IC50 of 10-6 M. Tetraphenyl hexyl bisphosphate revealed two binding sites with IC50’s of 10-4 M and 10-7 M, respectively. Understanding how these inhibitors interact with the enzyme will lead to better design of other inhibitors. Using Lineweaver-Burk plots, tetraethyl hexyl bisphosphate at different concentrations showed mixed inhibition while tetrabutyl hexyl bisphosphate showed competitive inhibition. Low concentrations of tetraphenyl hexyl bisphosphate also showed competitive inhibition of BuChE. Conformational changes of BuChE induced by these bisphosphates were analyzed using circular dichroism and tryptophan fluorescence. Circular dichroism showed no significant changes in conformation. However, using tryptophan fluorescence, there were observable changes in conformation with tetrabutyl hexyl bisphosphate and tetraphenyl hexyl bisphosphate. The degree of quenching correlates with inhibitory activity. Preliminary studies of beta-amyloid formation in vitro have shown that the bisphosphates prevent aggregation of amyloid beta-peptide 1-40. Based on these findings, we believe that bisphosphates are potential candidates for treating the symptoms of AD. Support in part by CSULB Minigrant Program and CSUPERB.
Poster #: 264
Campus: CSU San Bernardino
Poster Category: Other
Keywords: social interaction, attachment, oxytocin
Project Title: Oxytocin administration differentially affects maternal attachment in Sprague-Dawley and Fisher 344
Author List:
Aldersen, Erin; Graduate, Psychology, California State University, San Bernardino, Presenting Author
Humphrey, Danielle; Graduate, psychology, California State University, San Bernardino
Teran, Angie; Graduate, Psychology, California State University, San Bernardino, Presenting Author
Harmony, Zachary; Graduate, psychology, California State University, San Bernardino
Abstract: Fischer 344 (F344) rats exhibit reduced play behavior when compared to various other rat strains, including Sprague-Dawley (SD) rats. This suggests that F344 rats may be useful for studying social interaction deficits that are common in developmental psychiatric disorders. The purpose of the present study was to determine whether F344 and SD rats differ in their ability to form maternal attachment bonds during the preweanling period. In addition, we assessed whether the prosocial hormone oxytocin would alter attachment behavior in either rat strain. We measured attachment in F344 and SD rat pups on postnatal day (PD) 12 by using a maternal odor-induced conditioned place preference (CPP) procedure. The CPP procedure consisted of a habituation day, a conditioning day, and a test day, and was conducted in a Plexiglas three-compartment chamber. On the habituation day (PD 10), rats were given free access to the testing chamber for 1 min. On the conditioning day (PD 11), rats were given three 30-min conditioning sessions. Sessions consisted of either placing the pups in a holding cage with a lemon-scented dam (maternal group) or in an identical cage with lemon-scented cotton balls (neutral group). Conditioning sessions were separated by 3 h. On the test day (PD 12), all rat pups were given 5 min free access to the testing chamber, in which one side contained lemon scent and the other side was unscented. In Experiment 2, the identical procedures were used, with the exception that rat pups were injected with saline or oxytocin (250, 500, or 1000 ng, IC) prior to the first conditioning trial. In both experiments, time spent in the two compartments and the number of compartment entries were measured. As expected, rat pups in the maternal group spent more time in the lemon-scented compartment than rats in the neutral group. In the second experiment, SD rats were unaffected by oxytocin treatment, while F344 rats were impacted by oxytocin in a sex-dependent manner. Specifically, 250 ng oxytocin increased the odor preference of male F344 pups, while 1000 ng oxytocin increased odor preference in female F344 pups. The lower dose of oxytocin caused a general increase in locomotor activity (i.e., increased compartment entrees), but only in female F344 rats. In sum, these data indicate that SD and F344 rat pups exhibit a similar preference for dam-associated odors, but the preference shown by F344 rats could be enhanced by an exogenous application of oxytocin.
Poster #: 265
Campus: Cal Poly San Luis Obispo
Poster Category: Other
Keywords: Regeneration, Stem Cells, Ascidian
Project Title: Investigation of Blood Cell Populations in the Development and Regeneration of a Colonial Ascidian
Author List:
Boyd, Megan; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Balde, Erick; Undergraduate, Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo, Presenting Author
Medina, Elida; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Abstract: Botrylloides violaceus is a colonial marine ascidian capable of undergoing whole body regeneration after removal of all tissues except the peripheral vasculature. As chordates, colonial ascidians are more closely related to vertebrates than other model organisms for whole body regeneration. Evidence suggests that one or more populations of stem cells are present in the circulating blood of B. violaceus; these cells are identified as hemoblasts and may also be involved in asexual reproduction via budding. Analysis of blood cell frequencies during development and regeneration will reveal whether changes in the stem cell population are associated with these developmental changes. Fragments of B. violaceus were collected at a local dock, blood was drawn with a fine-gauge needle, and cells were quantified based on morphology using brightfield microscopy . Larvae released by the collected adult colonies were settled on glass slides to metamorphose into young colonies for blood sampling and for triggering regeneration by removal of body structures under a dissecting microscope. Comparisons using unequal variances t-tests suggest that hemoblasts are present in significantly greater abundance in young B. violaceus colonies and in colonies regenerating for 3, 6, and 9 days compared to in adults (p=0.0000024, p=0.00083, p=0.00022, p=0.0027). Hemoblast composition of B. violaceus is high in young colonies, but then decreases and stabilizes in regenerating colonies. This high percentage of hemoblasts may be associated with the readiness of B. violaceus to undergo regeneration at early stages in its life cycle. Currently, we are comparing blood composition in Botrylloides with the closely related ascidian genus Botryllus. Botrylloides regenerates more readily than Botryllus and a comparison may reveal whether the difference in regulation is related in part to a difference in standing stem cell populations.
Poster #: 266
Campus: Sacramento State University
Poster Category: Other
Keywords: Drosophila melanogaster, autism , neurodevelopment
Project Title: Developing Drosophila melanogaster as a model for the identification of environmental chemicals that confer risk to autism
Author List:
Nguyen, Kimberly ; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Murphy, Lillian ; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Trafton, Brandon; Undergraduate, Biological Sciences, California State University, Sacramento
Hindi, Zaed; Undergraduate, Biological Sciences, California State University, Sacramento
Ghenta, Kristina; Undergraduate, Biological Sciences, California State University, Sacramento
Lucich, Allicia ; , Biological Sciences, California State University, Sacramento
Torres, Christian; Undergraduate, Biological Sciences, California State University, Sacramento
Sidhu, Harjinder; Undergraduate, Biological Sciences, California State University, Sacramento
Mulligan, Kimberly; Biological Sciences, California State University, Sacramento
Abstract: Mounting evidence indicates that the interaction of environmental chemicals with specific genetic susceptibilities is linked to autism spectrum disorder (ASD), a condition that afflicts 1 of every 68 children in the United States. Yet, identification of specific environmental chemicals that interact with genes to cause ASD remains a critical gap in our understanding of ASD etiology. Given that 2,000 new chemicals are introduced each year with little to no toxicological data, the field desperately needs an efficient method for screening chemicals to identify those that confer risk to ASD. This project involves the development of assays using the common fruit fly, Drosophila melanogaster, for the rapid identification of chemicals that molecularly converge with fmr1 (fragile X mental retardation 1). Mutations in fmr1 are the most common single gene cause of ASD and the role fmr1 plays in neurodevelopment is conserved from flies to humans. Data from vertebrate model organisms suggest that gestational exposure to environmental chemicals, like polychlorinated biphenyl 95 (PCB-95), can significantly increase the risk of ASD in humans with mutations in fmr1. METHODS & RESULTS: We exposed embryonic and larval stage fruit flies with and without mutations in Drosophila fmr1 (dfmr1) to PCB-95. We used the courtship assay, a well-established paradigm for behavioral analysis of fruit flies, to determine that exposure to nanomolar concentrations of PCB-95 significantly decreases the courtship index (CI; a quantitative measure of courtship behaviors) in both wild-type (wt) and dfmr1 mutant flies, reflecting the ability of PCB-95 to impair the neurodevelopmental program. We also found that PCB-95 exposure more significantly lowers the CI of dfmr1 flies compared to wt flies, indicating that fmr1 confers susceptibility to PCB-95 in Drosophila. This result is consistent with findings in vertebrate model organisms and, thus, supports the use of Drosophila for chemical screening to identify those that enhance fmr1- associated deficits to increase the risk of ASD. To complement behavioral analysis, we are establishing an immunohistochemical assay to examine axon-pathfinding defects in an adult neural structure called the mushroom body (MB). We have determined that PCB-95 exposure increases axon pathfinding defects in the MB of wt flies. We are currently investigating if the severity of this defect is enhanced in dfmr1 mutant flies. Funding: CSUPERB New Investigator Award
Poster #: 267
Campus: CSU Stanislaus
Poster Category: Other
Keywords: cigarette smoke exposure, angiogenesis, gene expression, ,
Project Title: The Effect of Cigarette Smoke Exposure on Angiogenic Gene Expression in Chick Embryos
Author List:
Garcia, Sandra; Undergraduate, Biological Sciences, California State University, Stanislaus, Presenting Author
Bassi, Mandip; Undergraduate, Biological Sciences, California State University, Stanislaus, Presenting Author
Diaz, Celeste; Undergraduate, Biological Sciences, California State University, Stanislaus
Sosa, Sheila; Undergraduate, Biological Sciences, California State University, Stanislaus
Mahloch, Alexis; Undergraduate, Biological Sciences, California State University, Stanislaus
Thao, My Lo; Biological Sciences, California State University, Stanislaus
An, JungHa; Mathematics, California State University, Stanislaus
Grobner, Mark; Biological Sciences, California State University, Stanislaus
Abstract: Nicotine from cigarette smoke has been shown to affect the expression of genes involved in both blood vessel formation (neovascularization) and proliferation (angiogenesis). Genes that regulate angiogenesis include integrin 5 alpha, fibronectin, and vascular endothelial growth factor (VEGF). The objective of this study was to determine the effect of cigarette smoke on growth and angiogenesis in chorioallantoic membranes (CAMs) of chick embryos. CAMs were separated into control and experimental groups. The experimental groups were exposed to 5 mL or 15 mL of cigarette smoke for 6 days. CAMs were harvested after 2, 4, or 6 days of exposure to first-hand cigarette smoke, then total RNA was extracted using the Qiagen RNeasy Protect Mini Kit, and RNA concentration was determined. The mRNA was reverse transcribed to cDNA and gene expression was examined using QPCR and TaqMan probes. Results showed that blood vessel formation and proliferation in the CAMs exposed to cigarette smoke was greater when compared to the control. This is supported by the up-regulation of the genes involved in angiogenesis.
Acknowledgment: ORSP (CSU, Stanislaus), CVMSA (CSU, Stanislaus), LSAMP (CSU, Stanislaus)
Poster #: 268
Campus: CSU Fresno
Poster Category: Other
Keywords: 2,3-dehydrosilibinin, chemical modification, prostate cancer
Project Title: Synthesis and Antiproliferative Evaluation of 3-O-Aminoalkyl-5,7,20-O-trimethyl-2,3-dehydrosilibinins in Prostate Cancer Cell Models
Author List:
Vue, Bao; Department of Chemistry, California State University, Fresno, Presenting Author
Vignaue, Andre; Undergraduate, Department of Chemistry, California State University, Fresno, Presenting Author
Chen, Guanglin; Staff, Department of Chemistry, California State University, Fresno
Chen, Qiao-Hong; Department of Chemistry, California State University, Fresno
Abstract: Castration resistant prostate cancer is the cause of death to approximately 300,000 men each year worldwide, whereby, 28,000 are American men. Silibinin is the major chemical component of the seeds of milk thistle (silybum marianum). The crude extract of milk thistle (silymarin) has long been used as treatment for hepatotoxicity caused by poisoning mushroom and oxidative xenobiotics in European and Asian. Numerous cell-based and animal studies have demonstrated that silibinin and silymarin exhibit anti-prostate cancer potential. However, its clinical development has been limited by its moderate potency and suboptimal pharmacokinetics profile. Recently, 2,3-dehydrosilibinin has been reported by us and others to serve as a better lead compound than silibinin. The ultimate goal of the ongoing 2,3-dehydrosilibinin project in our research group is to engineer new derivatives with enhanced potency and bioavailability through appropriate structure manipulations for the treatment of prostate cancer. As part of this project, this study aims to explore the effect of incorporation of a nitrogen-containing group to 3-OH of 2,3-dehydrosilibinin through a three-carbon to five-carbon linker on the potency in three prostate cancer cell models. To this end, a four-step synthetic procedure, including trimethylation, oxidation, O-alkylation, and N-alkylation, towards the synthesis of 3-O-aminoalkyl-5,7,20-O-trimethyl-2,3-dehydrosilibinins has been successfully developed. Three target derivatives have been synthesized from silibinin through this synthetic sequence. Their structures have been fully characterized by interpreting the NMR data. Their anti-proliferative activity has been assessed in androgen-sensitive and androgen-insensitive prostate cancer cell lines using WST-1 cell proliferation assay. The results show that all these three derivatives have greater potency than silibinin and 2,3-dehydrosilibinin. We can conclude from these data that chemical modification at 3-OH of 2,3-dehydrosilibinin by incorporating a nitrogen-containing group is a vital strategy to optimize silibinin as anti-prostate cancer agents.
Poster #: 269
Campus: San Francisco State University
Poster Category: Other
Keywords: marine natural prodcuts chemistry, actinomycetes, anticancer drugs
Project Title: Two Novel Cytotoxic Naphthoquinone-Meroterpenoids Produced by Marine Sediment-Derived Streptomyces CP55-76
Author List:
Campit, Scott; Undergraduate, Chemistry and Biology, San Francisco State University, Presenting Author
Bray, Walter; Department of Chemistry and Biochemistry, UC Santa Cruz
Lokey, R.; Department of Chemistry and Biochemistry, UC Santa Cruz
Valeriote, Frederick; Josephine Ford Cancer Center
Amagata, Taro ; Chemistry and Biochemistry, San Francisco State University
Abstract: Actinomycetes are gram positive bacteria known for producing large arrays of bioactive secondary metabolites – organic compounds that possess several biological activities such as antioxidants, antibiotics and antitumor agents. This study explores the chemical diversity of marine sediment-derived Streptomyces CP55-76 to discover new solid tumor cytotoxins. Within our actinomycetes extract library, CP55-76 demonstrated significant prostate cancer cell line (LNCaP clone FGC) cytotoxicity using HeLa cell-based cytological profiling and in vitro cancer cell disk diffusion assay (CP-DDA). The One Strain Many Compounds (OSMAC) method was used to determine the seawater medium composition for the 10 liter cultivation of CP55-76. Based on CP-DDA guided fractionation, two new naphthoquinone-meroterpenoids and five known napyradiomycins were isolated. We determined the structures of these compounds using comprehensive one- and two- dimensional nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectroscopy (HRMS). Using CP-DDA as a tool for anticancer drug discovery, this unique cancer screening program identified the solid tumor selectivity and cellular targets of these compounds and led to the discovery of two new cytotoxins from CP55-76.
Poster #: 270
Campus: CSU San Bernardino
Poster Category: Other
Keywords: SSRI, western blotting, Sprague-Dawley rats
Project Title: Effect of SSRI expsoure on hippocampal BDNF functioning in adolescent rats
Author List:
Dhargalkar, Janhavi; Undergraduate, Psychology and Chemistry, California State University, San Bernardino, Presenting Author, Nagel Award Finalist
Rudberg, Krista; Graduate, Psychology, California State University, San Bernardino
Solis, Alexis; Undergraduate, Psychology, California State University, San Bernardino, Presenting Author
Harmony, Zachary ; Graduate, Psychology, California State University, San Bernardino
Abstract: Selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed class of antidepressant drugs, largely because of their effectiveness and favorable side-effect profiles. Unfortunately, the use of SSRIs in pediatric populations is limited due to reduced efficacy and their tendency to induce suicidal ideation in adolescents. Recently, we found that repeated treatment with fluoxetine and paroxetine caused increased anxiety-like behaviors in adolescent rats, as measured on the elevated plus maze and light/dark box. Moreover, adolescent rats did not show the adult-typical response of decreased serotonin utilization after repeated paroxetine treatment. The purpose of the present study was to determine if changes in brain derived neurotropic factor (BDNF) functioning are responsible for mediating these age-dependent behavioral and neurochemical effects. The rationale for this study was based on a growing body of evidence suggesting that the therapeutic effects of antidepressants are dependent on BDNF-mediated increases in neurogenesis. To test our hypothesis, we measured the expression of BDNF and the BDNF receptor, TrkB, after repeated paroxetine and fluoxetine treatment. Male and female adolescent Sprague-Dawley rats (n=6-7) were injected with paroxetine (2.5 or 10 mg/kg), fluoxetine (5 or 10 mg/kg), or vehicle for 10 consecutive days starting on postnatal day (PD) 35. On PD 45, the hippocampus of each rat was removed and then assayed for BDNF and TrkB expression using western blotting. In both male and females rats, BDNF expression was decreased after fluoxetine (5 and 10 mg/kg) treatment. Paroxetine (10 mg/kg) also decreased BDNF levels, but only in male rats. In contrast, TrkB expression was increased after SSRI treatment; however, this increase was only significant for male rats treated with the high dose of fluoxetine (10 mg/kg). In summary, repeated treatment with the SSRIs paroxetine and fluoxetine led to decreased BDNF expression in adolescent rats. This reduction in BDNF levels may be responsible for the reduced efficacy of SSRIs during adolescence. Interestingly, paroxetine had a greater effect on the BDNF functioning of male rats than females.
Poster #: 271
Campus: CSU Fresno
Poster Category: Programmatic (Core, Stem Cell, Bridges, PSM)
Keywords: undergraduate, medical, physics
Project Title: Tradition, innovation, and challenges in an undergraduate Biomedical Physics program at the California State University, Fresno
Author List:
Gherase, Mihai; Physics, California State University, Fresno, Presenting Author
Huda, Amir; Physics, California State University, Fresno
Abstract: For over half of century medical physics practice and research has changed medicine with discoveries and improvements in both the diagnostic imaging and the therapeutic modalities available. A certified clinical medical physicist is a highly lucrative career with starting average salaries exceeding $100,000/year based on a 2014 survey from the American Association of Physicists in Medicine. The increasing job demand saw a rapid increase in the number of graduate programs and students in this field. Concerns about the quality of educational programs resulted in the creation of the Commission on Accreditation of Medical Physics Educational Programs (CAMPEP) in 1994. Since 2014 the American Board of Radiology professional certification examinations has required CAMPEP certifications of both the graduate and the residency education of an aspiring candidate. Combined with an insufficient number of residency training programs in the United States, the admission into the graduate programs and residencies has become increasingly competitive. Initiated by one of us (AH) over a decade ago with funding from a National Institutes of Health grant, our undergraduate Biomedical Physics program was designed to offer its graduates a distinct educational advantage when applying for admission into graduate Medical Physics programs or tap into related careers that do not require graduate education. The program combines the traditional undergraduate Physics curriculum in the first two years with five required and one elective upper-division Medical Physics courses in the remaining two years. An important innovative element which sets our program apart from graduate programs, was the inclusion of laboratory components in the upper-division courses. Using traditional step-by-step guided and inquiry-based formats, the lab activities were possible through a unique collaboration with the local Veterans Affairs hospital. They seek to strengthen conceptual understanding, gain familiarity with clinical equipment through hands-on experience, and learning of experimental data analysis methods. The challenges faced were to provide a rigorous foundational knowledge by using student-engaging instruction and careful dissemination of material typically taught in graduate-level courses. A total of 26 students graduated since 2008; 11 students were admitted into graduate programs. Graduates entered careers in industrial research, clinical medical physics, and radiation safety.
Poster #: 272
Campus: CSU Northridge
Poster Category: Programmatic (Core, Stem Cell, Bridges, PSM)
Keywords: gene therapy, genome editing, disease model
Project Title: Creating Sickle-Cell Disease and Beta-Thalassemia Cell Lines to Facilitate Testing of Gene Therapy Approaches for Hemoglobinopathies
Author List:
Parma, Marie Eloise; Graduate, Biology, California State University, Northridge, Presenting Author
Garcia, Zulema; Department of Microbiology, Immunology and Molecular Genetics, UCLA
Kohn, Donald; Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, UCLA
Abstract: Current therapies to treat monogenic hemoglobinopathies, such as sickle cell disease (SCD) and beta-thalassemia (B-thal), are non-curative with the exception of allogeneic bone marrow transplant (BMT). However, BMT is associated with multiple immunological complications and HLA-matched donor availability is limited. Gene therapy approaches offer viable alternatives to allogeneic BMT and target the ex-vivo modification of a patient’s own hematopoietic stem cells for autologous transplant. A therapeutic effect can be achieved via the introduction of a globin-expressing lentiviral vector or targeted gene editing technologies, yet we still need to improve safety and delivery efficacy. The development of an immortalized human erythroid progenitor cell line with the appropriate genotype could facilitate testing gene therapy approaches. We have generated a B-thal cell line via zinc finger nuclease (ZFN) mediated site-directed knockout of the beta hemoglobin gene in CD34+ human umbilical cord-derived erythroid progenitor cells (HUDEP). We used a HUDEP-2 cell line that terminally differentiates into mature erythrocytes and is capable of producing adult hemoglobin. Additionally, we generated a SCD cell by co-delivering an oligonucleotide donor (oligo) carrying the sickle mutation and site-specific ZFNs that target the beta hemoglobin locus. We have exploited normal cellular DNA repair pathways to generate the two cell lines: the B-thal cell line can be generated via non-homologous end joining and the SCD cell line via homology-directed repair. We monitored allelic disruption via Surveyor nuclease assay and found 20 % ZFN-induced DNA cutting at the target site in the pool samples. In the ZFN-oligo treated pool samples, we measured 10 % of gene modification by conducting a qPCR-based assay that quantifies a co-introduced HhaI base change frequency. We have then performed single cell sorting and clonal expansion of the modified HUDEP-2 cells. We selected clones that were homozygous for the desired modification and results were validated by Sanger sequencing with single nucleotide polymorphism analysis. We additionally conducted high performance liquid chromatography to compare the globin chains produced by the clones and our preliminary findings revealed that our SCD cell lines produced sickle hemoglobin even without differentiation. Interestingly, our B-thal cell lines lacked the production of beta hemoglobin but showed increased levels of fetal hemoglobin.
Poster #: 273
Campus: Cal Poly San Luis Obispo
Poster Category: Programmatic (Core, Stem Cell, Bridges, PSM)
Keywords: froce production, microcirculation, cardiovascular disease
Project Title: Calf Force Production as a Measure of Vessel Remodeling and Vasodilation in a C57 Bl/6 Ischemic Mouse Model
Author List:
Magill, Eric; Graduate, BMED, California Polytechnic State University, San Luis Obispo, Presenting Author
Cardinal, Trevor; BMED, California Polytechnic State University, San Luis Obispo
Abstract: Cardiovascular disease continues to be the leading cause of death in the United States. In 2010, it accounted for 30% of deaths worldwide and costed the global economy $863 billion. Narrowing or blockage due to plaque buildup or atherosclerosis in a blood vessel is the most prevalent cause of cardiovascular disease. The ability for vessels to remodel after occlusion can affect patient recovery and outcome. Previously, our lab found anterior gracilis collateral remodeling and vasodilation to be the same in male and female C57 Bl/6 mice via in vivo imaging and whole mount histology. Clinically however, evaluation techniques such as pain free walking distance, a measure of downstream muscle function, are used. The goal of this project is to develop an evaluation method, calf muscle force production, which better translates this research to the clinic. It is hypothesized there is no difference in force production between male and female C57 Bl/6 mice because remodeling and vasodilation in the anterior gracilis collateral are equivalent. The left femoral artery of male and female (n=6,6) C57 Bl/6 mice was ligated distal to the profunda femoris and proximal to the popliteal branch. Sham surgery was performed on the contralateral limb to serve as a surgical control. Day 7 post-ligation, twitch force, tetanus, fatigue, and recovery were measured in the calf muscles of both hindlimbs using in vivo force production. Force output was measured by a transducer attached to the Achilles tendon after electrically stimulating the calf to contract via the sciatic nerve. Specific force was calculated by normalizing the data using dry muscle weight. Females exhibited impaired specific force during maximum twitch (p<0.05), 4.44±0.25 (mean±SE) vs 2.50±0.42 mN/mg (sham vs ligated); and tetanus (p<0.05), 15.19±2.45 vs 6.67±1.05 mN/mg (sham vs ligated), at 75 Hz. Male specific force was not impaired during maximum twitch, 2.86±0.26 vs 2.63±0.31 mN/mg (sham vs ligated); or tetanus, 11.45±1.50 vs 8.77±1.51 mN/mg (sham vs ligated), at 75 Hz. Neither sex showed impairment during fatigue or recovery. This marked impairment in females, not present in males, indicates a female deficiency in remodeling or vasodilation of chronic but not acute downstream vessels to the ligation site. Along with establishing a novel method of in vivo calf force measurement, this study gives insight into why treatments in the area of cardiovascular research do not often translate to clinical success.
Poster #: 274
Campus: CSU Fullerton
Poster Category: Proteins (Include Proteomics)
Keywords: membrane topology, fluorescence microscopy, metal transporter
Project Title: Defining the membrane topology of transmembrane (TMEM)-163 protein
Author List:
Nino, Katia; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Torres, Kyelo; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Rivas, Tiffany; Undergraduate, Biological Science, California State University, Fullerton
Cuajungco, Math; Biological Science, California State University, Fullerton
Abstract: We previously identified the TMEM163 protein through a yeast two-hybrid screen in search of interaction partners for the Transient Receptor Potential Mucolipin-1 ion channel. TMEM163, also known as synaptic vesicle 31 (SV31) was first identified in rat brain synaptosomes using a proteomic screen. It was reported to have a predicted six transmembrane (TM) domains with amino (N)- and carboxyl (C)-termini facing the cytoplasmic side of the plasma membrane. Although the function of TMEM163 remains to be fully elucidated, a previous report showed that it binds zinc, nickel, and to a lesser extent, copper. Investigations in our laboratory revealed that TMEM163 could be a zinc transporter; however, our bioinformatics analysis showed that the predicted membrane topology of TMEM163 could either have the N- and C-termini facing intracellularly or extracellularly. We hypothesize that N- and C-termini of TMEM163 are located on the extracellular side based on our preliminary observation that it transports zinc into the cells. To this end, we performed site directed mutagenesis (SDM) to systematically insert a hemagglutinin (HA) peptide tag between each transmembrane domain (TM1-TM6), and either at the N- or C-terminus end of the protein. We then transfected human embryonic kidney (HEK)-293 cells with each corresponding TMEM163 clone containing inter-TM HA tag and a control vector. Using immuno-cytochemistry (ICC), two parallel trials of transfected cells were either permeabilized with 0.5% Triton X-100 detergent, or non-permeabilized. The cells were then incubated with a mouse anti-HA primary antibody, followed by anti-mouse secondary antibody conjugated with an Alexa-488 fluorophore. The images were then analyzed using fluorescence microscopy. We found that TMEM163 tagged with the HA peptide at the N- or C-terminus suggest an extracellular orientation. Furthermore, ICC results using TMEM163 with HA tag between TM1-TM2 or TM2-TM3 also suggest an extracellular orientation. Overall, our preliminary data suggest that the membrane topology of TMEM163 confer extracellular N- and C-termini, which contradicts the published predicted protein topology in the literature. Additional investigations to validate the current observation will include the use of different peptide tags using ICC. Knowledge from this study could open new research avenues on the structure and function of TMEM163 in zinc transport.
This work was funded by the CSUPERB Research and Development Grant 2016.
Poster #: 275
Campus: CSU San Marcos
Poster Category: Proteins (Include Proteomics)
Keywords: Curli, Amyloid, Fluorescence Quenching
Project Title: The Terminal Regions of CsgF may be Involved in Binding Protein Ligands
Author List:
Aranda, Isamar; Undergraduate, Chemistry and Biochemistry, California State University San Marcos, Presenting Author
Guerrero, Karen; Undergraduate, Cheristry and Biochemistry, California State University San Marcos, Presenting Author
De Morais, Rafael Lima; Undergraduate, Chemistry and Biochemistry, California State University San Marcos
Assuncao, Davi; Undergraduate, Chemistry and Biochemistry, California State University San Marcos
Souza Marimon, Patricia; Undergraduate, Chemistry and Biochemistry, California State University San Marcos
Alshulah, Maryam; Graduate, Chemistry and Biochemistry, California State University San Marcos
Jayasinghe, Sajith; Chemistry and Biochemistry, California State University San Marcos
Abstract: Introduction: Bacteria, such as E.coli and Salmonella, contain hair-like cell surface organelles termed curli. Curli serve as adhesins, mediating interactions between bacterial and host cells that are essential for host cell colonization, and play an important role in the pathogenesis of bacterial disease. Curli assembly involves six proteins, CsgA, CsgB, CsgC, CsgE, CsgF, and CsgG. CsgE and CsgF are thought to act as chaperones to help prevent the premature aggregation of CsgA and CsgB, and to help transport these proteins to the cell surface, where they are assembled to form curli. We have observed that CsgF is able to inhibit the aggregation human islet amyloid polypeptide (hIAPP), an amyloidogenic polypeptide that is unrelated to curli. We sought to determine the nature of any CsgF-hIAPP interaction using four cysteine mutants of CsgF labeled with the fluorescent probe IAEDANS. Methods: BL21 bacterial expression system was used to express four different mutants of CsgF (23C, 66C, 97C and 123C where the wild type residue at the respective position was replaced by a cysteine). The protein was purified using Ni affinity chromatography. The cysteines were labeled using IAEDANS and purified again through Ni affinity chromatography and a PD-10 column. Fluorescence spectroscopy was used to determine the ability of CsgF to interact with the non-aggregating rat Islet Amyloid Polypeptide (rIAPP). Results and Discussion: In the presence of rIAPP the four IAEDANS labeled CsgF proteins exhibited emission maxima of 477, 475, 480, and 472 nm respectively. In the presence of rIAPP the emission maxima of IAEDANS at positions 66 and 123 showed significant blue shifts (of 13 and 17 nm respectively) suggesting that these positions are involved in the interaction with rIAPP. We measured quenching of IAEDANS by acrylamide in the absence and presence of rIAPP. In the absence of rIAPP IAEDANS labeled at positions 23, 66, 97, and 123 gave Stern Volmer quenching constants of 11.1, 10.1, 12.7, and 18.8 respectively, while in the presence of rIAPP these values were 8.3, 16.1, 18.5, and 8.6. This reduction in the quenching constant for position 123 in the presence of rIAPP suggests that the fluorophore at this position becomes protected from quenching. The protection could either be due to a conformational change in CsgF driven by binding rIAPP or could be due to residue 123 interacting directly with rIAPP.
Poster #: 276
Campus: CSU Northridge
Poster Category: Proteins (Include Proteomics)
Keywords: Trk receptors, NMR spectroscopy, protein dynamics
Project Title: Localized motions of residues in TrkB-d5 point to a role for dynamics in binding selectivity and allostery
Author List:
Surinarintr, Akachai; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Crowhurst, Karin; Chemistry and Biochemistry, California State University, Northridge
Abstract: Neuronal growth and survival are mediated by the interactions of Tropomyosin-related kinase (Trk) receptors and neurotrophins (NTs). Activation of TrkB occurs when a NT binds extracellular domain 5 of TrkB (TrkB-d5); this leads to intracellular autophosphorylation and activation of its associated signaling pathways. Misregulation of the NT-Trk interactions can lead to neurodegenerative diseases such as Alzheimer’s or Parkinson’s. There is evidence that TrkB-d5 undergoes conformational changes when binding a NT, however, molecular characterization of the TrkB-d5 / NT interaction is not well defined. This conformational change may play a role in Trk selectivity for a particular NT and in determining which signaling pathway is ultimately activated. We hypothesize that the changes in backbone and side chain dynamics (motions) of TrkB-d5 are essential to binding selectivity. Nuclear Magnetic Resonance (NMR) spectroscopy is ideal for investigating the conformational changes and the timescale during which motions take place in TrkB-d5. Studying TrkB-d5 by NMR will help us glean a greater understanding of the role dynamics play in the binding selectivity of Trk receptors and their related NTs.
We previously assigned the sidechain chemical shifts of TrkB-d5 using a novel mutagenesis approach. With the chemical shift assignments, we are now able to investigate the dynamics of TrkB-d5. Our dynamics data on apo TrkB-d5 (not bound to NT) indicate that key residues within the binding interface and a cluster of residues remote to the interface have us-ms timescale motions, which is consistent with the timescale at which folding and allosteric regulation frequently occur in proteins. Results from hydrogen-deuterium exchange experiments showed that the core of TrkB-d5 is very well protected from solvent. The exchange data indicate that the amide backbone of the core is forming hydrogen bonds and is well structured, despite evidence for dynamics nearby. We are currently working on obtaining dynamics data and hydrogen/deuterium exchange data for hTrkB-d5 complexed to NT4. Comparing data from unbound and NT-bound TrkB-d5 will help us better understand the allosteric properties of Trk and provide key information to help others design targeted therapeutics for neurodegenerative diseases.
We gratefully acknowledge the NIH for research support (SC2-GM094127) and the NSF for funding the purchase of our NMR spectrometer (CHE-1040134).
Poster #: 277
Campus: Sacramento State University
Poster Category: Proteins (Include Proteomics)
Keywords: apolipoprotein, amyloid, protein folding
Project Title: Acidic pH promotes amyloid formation in full-length apolipoprotein A-I and its amyloidogenic mutants
Author List:
Lee, Tim ; Undergraduate, Chemistry, California State University, Sacramento, Presenting Author
Nguyen, Nancy; Undergraduate, Chemistry, California State University, Sacramento, Presenting Author
Roberts, Linda; Chemistry, California State University, Sacramento
Abstract: Apolipoprotein A-I (apoA-I), a major component of high-density lipoprotein (HDL) found in human plasma, contains many anti-atherogenic properties and is correlated with lowered risk for heart disease. However, misfolding of apoA-I, either due to mutation or environmental factors, contributes to amyloid formation in various tissues and organs, including the heart. Although it has been shown that acidic pH can influence the structure of an N-terminal fragment of apoA-I commonly found in amyloid deposits, the effect of pH and other factors such as ionic strength on aggregation of full-length apoA-I has not been investigated. The goal of this research was to determine how variation in physiological conditions like pH and salt concentration impact the rate of amyloid formation in apoA-I and its amyloidogenic mutants G26R and L178H. Purified apoA-I protein was combined with McIlvaine’s buffer to give a final concentration of 0.1 mg/mL protein and pH ranging from 2 to 7. Aggregation was detected by light scattering intensity at 340 nm and amyloid aggregation was detected by binding to Thioflavin T (ThT). All three proteins aggregated extensively between pH 4 and 5, with the highest level occuring at either pH 4 or 4.5. The isoelectric point of apoA-I is about 4, which suggests that some of the aggregation could simply be due to isoelectric point precipitation. ThT binding corrected for light scattering was the highest in the pH 4 and 4.5 samples, indicating the presence of amyloid structure. Isoelectric point precipitation should be prevented by high salt for ions low in the Hofmeister series, e.g. chloride ion. Addition of up to 1.0 M NaCl did not prevent aggregation at acidic pH. These results show that apoA-I and its amyloidogenic mutants are all capable of forming amyloid structure at acidic pH which may contribute to deposition of amyloid protein in acidic microenvironments in vivo.
Poster #: 278
Campus: CSU Fullerton
Poster Category: Proteins (Include Proteomics)
Keywords: Tetrahydromethanopterin, Methanosarcina mazei, MM1853
Project Title: Investigation of Electron Transfer Systems to Methanogen Protein MM1853, an Oxidoreductase Enzyme involved in Tetrahydromethanopterin Biosynthesis
Author List:
Pang, Chao; Graduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Gonzalez, Stephen; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Moscaira, Jose; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Rasche, Madeline; Chemistry and Biochemistry, California State University, Fullerton
Abstract: Methane production by archaea depends on the synthesis of tetrahydromethanopterin (H4MPT), a pterin-containing cofactor that carries one-carbon units. Previous investigators have hypothesized the existence of two redox reactions within the nine steps of H4MPT side chain biosynthesis. Dihydromethanopterin reductases are recognized to catalyze a redox reaction that is the ninth and last step of the pathway. However, the enzyme catalyzing the other proposed redox reaction has not yet been identified. Complementation studies in the methylotrophic bacterium Methylobacterium extorquens AM1 have indicated that the Orf19 protein is involved in H4MPT biosynthesis. The crystal structure of an Orf19 homolog (MM1853) in Methanosarcina mazei has revealed the presence of one flavin mononucleotide (FMN)-binding site per subunit, consistent with the activity of an oxidoreductase. Therefore, we propose that the protein MM1853 catalyzes the other redox reaction, which corresponds to the third step of H4MPT side chain biosynthesis. Identifying electron donors capable of reducing the FMN prosthetic group of MM1853 is a prerequisite to testing this hypothesis. Here we describe the purification and characterization of a six-histidine (His6)-tagged version of MM1853 and identification of electron donors that may serve as the reductant for an MM1853 enzyme assay. Native and SDS PAGE showed that purified His6-MM1853 occurred as a homodimer of 25-kDa subunits. The UV-visible spectrum of the purified protein showed absorbance peaks at 380 and 460 nm, characteristic of oxidized FMN. NADH and NADPH were incapable of directly reducing the flavin cofactor; however, 50 mM dithiothreitol eliminated the FMN peaks within 30 min with a rate of 1.33 µM/min, indicating successful electron transfer to MM1853. Interestingly an electron transfer system consisting of NADH and flavin-reducing enzyme (Fre) from Salmonella enterica could also reduce the FMN peaks of MM1853 at a rate of 8-20 µM/min. These results provide the basis for an oxidoreductase assay that will allow us to test the hypothesis that NADH, Fre, and MM1853 can reduce the ribose ring of dihydropterin β-ribofuranosylaminobenzene 5’-phosphate in the third step of H4MPT side chain biosynthesis. Development of this assay would provide the first biochemical evidence for the enzymatic role of MM1853 in H4MPT synthesis and establish MM1853 as a target for inhibiting the microbial production of methane as a greenhouse gas. (Funding: NSF)
Poster #: 279
Campus: CSU Fresno
Poster Category: Proteins (Include Proteomics)
Keywords: Clp, cyclic di-GMP, Xanthomonas species
Project Title: Clp senses cyclic di-GMP through its dimerization domain
Author List:
Curiel, Joel; Graduate, Biology, California State University, Fresno, Presenting Author
An, Dongkuk; Undergraduate, Biology, California State University, Fresno, Presenting Author
Gunasekara, Sanjiva
Youn, Hwan; Biology, California State University, Fresno
Abstract: CRP-like-protein (Clp) is a transcription factor found in Xanthomonas species that senses host plant cells and regulates the production of various virulence factors. In this way, Clp plays a master regulator role in causing Xanthomonas-derived plant diseases. When Xanthomonas species are outside of host plant cells, Clp is kept inactive by high levels of cyclic di-GMP. Biochemically, apoClp is active and becomes inactivated by binding to the ligand cyclic di-GMP. However, Clp does not have a clear cyclic di-GMP motif, thus the Clp region critical for the binding and responsiveness of cyclic di-GMP is not known. To identify the binding region, we adopted a chimeric protein approach which fuses the N-terminal portion of CRP from Escherichia coli (a model protein) to the C-terminal portion of Clp. Four different chimeric proteins which differ only in their junction sites (Pro131, Phe157, Arg163 and Pro175; all Clp numbering) were made and their responses to cyclic di-GMP were tested. Since CRP is unable to respond to cyclic di-GMP, a perturbed cyclic di-GMP responsiveness from a chimeric protein would indicate that an important Clp residue for ligand sensing is compromised. When assayed in vivo, the first three chimeras (CRP-Pro131-Clp, CRP-Phe157-Clp, and CRP-Arg163-Clp) were indistinguishable from Clp. In contrast, when assayed with a saturated amount of cyclic di-GMP, the activity of CRP-Pro131-Clp was completely inhibited as was Clp, but the activities of the latter two chimeras were only partially inhibited. Our results suggest that the Clp region between Pro131 and Phe157, which comprises the dimerization domain, contains important residues for cyclic di-GMP binding. A sequence alignment shows that Clp and CRP differ in this region by 16 amino acids. Future plans include alanine scanning experiments at the 16 amino acid positions, which will narrow down which Clp residues (between Pro131 and Phe157) are particularly important for cyclic di-GMP binding and sensing.
Poster #: 280
Campus: CSU Northridge
Poster Category: Proteins (Include Proteomics)
Keywords: HdeA, NMR techniques, computational modeling
Project Title: Use of experimental and computational methods to model increased flexibility and N-terminal structure in the chaperone protein HdeA at pH 2.8
Author List:
Widjaja, Marlyn; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Crowhurst, Karin; Chemistry and Biochemistry, California State University, Northridge
Abstract: HdeA is a small chaperone protein that protects other proteins from acidic environments. It is expressed in the periplasm of several pathogenic bacteria, such as E. coli. At physiological pH, HdeA adopts a folded dimer conformation, which is inactive. At low pH, HdeA unfolds and dissociates into a disordered monomer. It utilizes exposed hydrophobic patches to bind other periplasmic proteins to prevent their unfolding and aggregation. This mechanism is key in the spread of dysentery as it helps pathogens survive in the acidic environment of our stomach.
Our group is interested in the mechanism by which HdeA is activated. We know that HdeA unfolds and dissociates into its active form between pH 3.0 and 2.0. Using atomic resolution NMR techniques, we seek to characterize the protein motions of HdeA in this transitional pH range. My project specifically focuses on pH 2.8. Earlier experiments showed that HdeA is still a folded dimer at pH 2.8, but its structure is looser and more flexible than observed at higher pH. Interestingly, we found that the usually dynamic N-terminus has a decreased in solvent accessibility at pH 2.8 compared to higher pH values, suggesting the presence of secondary structure. Though this observation is likely caused by transient structure formation, we hypothesized that the stronger interactions should still be observable experimentally and computationally. 15N NOESY NMR experiments, which probe through-space interactions between backbone amides, provided evidence that the flexible N-terminus may form stable interactions with residues in helix A, specifically between residues 7 and 8 in the N-terminus with residues 23 and 16, respectively. To better visualize and understand these results, we used the computer modeling software CS-Rosetta, which utilized algorithms and our NMR chemical shift values to produce an ensemble of structures that best fit the data. Within the results, we found a small population of conformations containing a short helix at the N-terminus (residues 5-8), which is also positioned close to helix A. This transient structure may be responsible for the observed decrease in solvent accessibility. Our ongoing research will continue to focus on this N-terminal region of HdeA at pH 2.8 to determine if the transient structure formation plays a role in chaperone activation.
We gratefully acknowledge the NIH for research support (SC3-GM116745) and the NSF for funding the purchase of our NMR spectrometer (CHE-1040134).
Poster #: 281
Campus: San Francisco State University
Poster Category: Synthetic Chemistry
Keywords: HDAC inhibitor, cis-stilbene, stereospecific synthesis
Project Title: Stereospecific synthesis of cis-stilbenes from benzaldehydes and phenylacetic acids via sequential Perkin condensation and decarboxylation
Author List:
Thompson, Clayton; Undergraduate, Chemistry and Biochemistry, San Francisco State University, Presenting Author, Nagel Award Finalist
Orellana, Matthew; Undergraduate, Chemistry and Biochemistry, San Francisco State University
Lloyd, Shannon; Undergraduate, Chemistry and Biochemistry, San Francisco State University
Wu, Weiming; Chemistry and Biochemistry, San Francisco State University
Abstract: Histone deacetylase (HDAC), one of the major enzymes involved in regulating gene expression, removes acetyl groups from lysine residues on histones to allow for tighter DNA wrapping. There are four main classes of HDACs ; the NAD dependent Class III HDACs hold promise as potential targets for cancer treatments. Two isoforms, SIRT1 and SIRT2 play key roles in cancer chemotherapy due to their involvement in tumorigenesis and cancer cell proliferation. This suggests that SIRT1/2 inhibition may lead to the discovery of new drug leads. Several biphenylcarboxylic acids were found to be excellent inhibitors of the yeast enzyme Sir2p (commonly used in inhibition studies). However, our calculations showed void in the binding of these molecules in the active site, indicating that increasing the distance between the two phenyl groups should further enhance the binding of these molecules with Sir2p. We have thus designed molecules with alkynyl and alkenyl groups separating the two phenyl groups and thus elongate the length of the molecules. In this project, we are developing efficient synthesis of stilbene derivatives as part of our overall goal of discovering potent inhibitors of HDACs. Stilbene derivatives also have many applications in industry as dyes, optical brighteners, phosphors, and scintillators. Stilbene moieties are also common in natural products such as resveratrol and pterostilbene. In this presentation, we report the successful stereospecific synthesis of 13 substituted cis-stilbenes in high yield from the sequential Perkin condensation and decarboxylation. The synthetic method combines the preparation of α-aryl-trans-cinnamic acids from the Perkin condensation of benaldehydes and phenylacetic acids and their cuprous oxide-catalyzed decarboxylation. The synthetic method allows the mix-and-match of different substituents on the two phenyl groups. Overall, the two-step sequence represents a convenient, stereospecific, flexible and high-yielding method for the synthesis of substituted cis-stilbenes. A manuscript describing the results has just been accepted for publication. We will continue our synthesis and investigation of these aromatic compounds as HDAC inhibitors.
We acknowledge the support of this project by NIH (Grant SC1 GM095419) and SFSU Office of Research and Sponsored Programs and the Department of Chemistry & Biochemistry.
Poster #: 282
Campus: CSU Bakersfield
Poster Category: Synthetic Chemistry
Keywords: peroxidation, 7-dehydrocholesterol, microwave synthesis
Project Title: Towards Understanding of Peroxidation of Mammalian Sterols: Microwave-Assisted Synthesis of 7-Dehydrocholesterol Isomers
Author List:
Olvera, Amanda; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Ramos Flores, Juan; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Muchalski, Hubert; Chemistry, California State University, Fresno
Abstract: Molecules of biological importance, such as polyunsaturated fatty acids (PUFAs) and sterols are primary targets for the H-atom abstraction by a peroxyl radical. This process, known in biological sciences as lipid peroxidation, receives a considerate amount of research attention from multiple fields.[1,2] There is a growing consensus that products of lipid peroxidation play a significant role in the pathophysiology of many human disorders. 7-Dehydrocholesterol (7-DHC) is a lipid that is the biosynthetic precursor to cholesterol and vitamin D3. This compound plays a crucial role in the human neurodevelopmental disorder, Smith—Lemli–Opitz Syndrome (SLOS). Characteristic to SLOS patients are increased levels of 7-DHC and lower than normal levels of cholesterol in the plasma. SLOS patients also suffer from birth defects and delayed mental development. The rate constant of peroxidation of 7-DHC is 200 times higher than that of cholesterol contributing to the growing amount of evidence that links SLOS to peroxidation of 7-DHC.[3,4] The overarching goal of this project is to better understand the relationship between the structure and oxidation rate in 7-DHC. Pyrocholecalciferol (1) and isopyrocholecalciferol (2) are C-9 and C-10 diastereomers of 7-DHC where the hydrogen atom that is attacked by the radical resides in the diametrically different steric environment. To make progress towards this goal, we developed a practical synthesis of pyrocholecalciferol (1) and isopyrocholecalciferol (2) by a thermal electrocyclic reaction of cholecalciferol (vitamin D3). Our preliminary attempts at the synthesis according to the literature procedure showed a low level of reproducibility and revealed that complete consumption of the starting material requires long reaction times (over 12 h). Also, the extended reaction time also caused significant decomposition.
Results: We developed a practical synthesis of sterols 1 and 2 using a microwave reactor. Contrary to our expectations, the starting material and the products were not sensitive to high temperatures. The new protocol significantly decreased the reaction time (from 12 h to 30 min) and reduced the amount of solvent used by 90%. Additionally, we observed almost undetectable levels of product decomposition despite high reaction temperature (200 °C).
Acknowledgments: This material is based upon work supported by the CSU-LSAMP program funded by NSF under grant #HRD-1302873, CSU Office of the Chancellor, and Fresno State.
Poster #: 283
Campus: Sacramento State University
Poster Category: Synthetic Chemistry
Keywords: dendrimer, glycodendrimer, organic synthesis
Project Title: Synthesis of an Octavalent Dendrimer for Glycodendrimer Synthesis
Author List:
Cerney, James; Undergraduate, Department of Chemistry, California State University, Sacramento, Presenting Author
McReynolds, Katherine; Department of Chemistry, California State University, Sacramento
Abstract: Dendrimers have been found to be useful tools among a variety of biomedical applications, including as transport molecules, scaffolds, and inhibitory agents. These globular molecules consist of a central core which is then branched using linker molecules. The number of functional ends increases with each branching step. Our research is based on the synthesis of glycodendrimers containing aminooxy linkages. By having a higher number of functional ends, the molecule is able to bind to an increased number of targets. The size of these molecules also plays an important role in their effectiveness through mimicking the size of the receptors normally targeted by infectious agents. Many commercially available dendrimers exhibit toxicity. Our research focuses on synthesizing molecules that have binding affinity to the desired biological targets with low toxicity.
Currently, work is being done to synthesize an aminooxy terminated octavalent core. The linker was first synthesized through a two-step process. First, one of the hydroxyl groups of ethylene glycol was attached to acrylonitrile in a yield of 43%. The other alcohol was then tosylated in a 74% yield. The tetravalent core was next synthesized through an SN2 reaction involving the central core molecule, 2,2’-oxybis(ethylamine), with the tosylated linker, resulting in a 38% yield. The tetra-nitrile core then underwent a two-step, one pot reaction in which the nitrile termini were reduced and Boc protected resulting in a 47% yield.
In future work, the tetravalent core will undergo deprotection revealing amine terminated ends. This will be followed by further branching, revealing an octavalent dendrimer core through a Michael addition using acrylonitrile. The nitrile ends will then undergo a reduction/ Boc-protection, followed by a deprotection of the Boc protected ends revealing the octa-amine core. The octa-amine core will then be reacted with a short linker revealing the aminooxy terminated core, which will then be ready for glycosylation.
Funding: NIH 1SC3GM119521-01
Poster #: 284
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Kinetic Resolution, Kinase Inhibition, Atropisomerism
Project Title: Atropisomeric Synthesis of Selective Kinase Inhibitors: A Scalable Solution to a Problem in Drug Discovery
Author List:
Cardenas, Mariel; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Dinh, Andrew; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Abstract: Atropisomerism is a form of chirality that is common throughout drug discovery, specifically in kinase inhibition. Atropisomerism differs from normal point chirality in that the chirality is about a bond (i.e. aryl-aryl bonds) and hence racemization can spontaneously occur via bond rotation. Most atropisomeric bioactives exist as rapidly interconverting mixtures of enantiomers. While these molecules may not appear chiral, they bind to the target active site in a single atropisomeric conformation, with the non-relevant atropisomer contributing little to the desired activity.
Pyrrolopyrimidines (PPYs) are known to be promiscuous tyrosine kinase inhibitors. We have recently demonstrated that atropisomerically stable analogs of these molecules possess improved target selectivity. Furthermore, we found that each atropisomeric enantiomer possessed activity towards different kinases, effectively decoupling the activity of the rapidly racemizing parent compound. One bottleneck of this project is our reliance on semi-preparative chiral HPLC separation to obtain enantiopure material, limiting the scalability and efficiency of our current syntheses. To address this, we have developed a nucleophilic kinetic resolution to obtain enantioenriched analogs without the use of chiral semi-preparative HPLC.
Kinetic resolution is achieved by addition of thiophenol into the PPY scaffold via nucleophilic aromatic substitution (SNAr), catalyzed by quinine-based chiral quaternary ammonium salts. Starting from racemic, atropisomerically stable PPY, we can enantioselectively convert just about 55.1% of the material to the SNAr product, leaving enantioenriched starting material in enantiomeric ratios of 97:3. We have successfully applied this strategy across a series of differentially substituted PPY analogs. Furthermore, we have extended this chemistry to include a range of nucleophiles including amines, azides, and heterocycles. This chemistry was also found to be amenable to diaryl anilines (the most common motif in kinase inhibition). To demonstrate the utility of this chemistry, we performed this kinetic resolution on preparative scales and have transformed the enantioenriched SNAr products and starting material to their respective enantioenriched final products for analysis in kinase inhibition assays.
Poster #: 285
Campus: Sacramento State University
Poster Category: Synthetic Chemistry
Keywords: electrochemistry, catalysis, organic synthesis
Project Title: Polymer Supported Ni-Salen Catalysts in Mediated Electrohydrocyclization Reactions
Author List:
Clendenin, Brittanie ; Graduate, Chemistry Department, California State University, Sacramento, Presenting Author
Miranda, James; Chemistry Department, California State University, Sacramento, Presenting Author
Abstract: The electrohydrocyclization (EHC) reaction to form carbocyclic rings have been used as the key step in the synthesis of several anticancer compounds and natural products. Metal-salen compounds can serve as excellent electrochemical mediators in these reactions, resulting in a chemoselective process due to the reaction being run at a more positive potential than unmediated reactions. In addition, since only a catalytic amount of mediator is needed, the resultant chemical waste is greatly reduced, resulting in a more environmentally friendly reaction. Previous work in our group studied these catalytic reactions experimentally and computationally.
We have developed a polymer supported Ni-salen compound that can serve as a catalyst in EHC reactions. Synthesis of a Ni-salen compound that can be anchored to a polystyrene resin was accomplished using a resin capture process. Several catalysts were synthesized and studied, both with and without the polystyrene resin. The primary benefit of this heterogeneous catalyst is the drastic improvement of reaction workup and purification. Infrared spectroscopy and atomic absorption were used to characterize the polymer supported catalysts.
Cyclic voltammetry showed that 5 mM Ni-salen gives a reversible wave at 1.6 V (vs Ag/AgCl). When 50 mM of the EHC substrate was added to the Ni-salen solution, the existence of a catalytic current was found to exist. The cyclic voltammograms were run using a glassy carbon working electrode and platinum wire counter electrode. The experiment was run in dimethylformamide (DMF) solvent using 0.1 M tetra-n-butylammonium hexafluorophosphate as the supporting electrolyte.
The cyclization reaction using nonpolymer supported catalysts were run first, and then compared to the results of the polymer supported catalyst. The reactions were run using EHC substrate,10 mmol % catalyst, dimethyl malonate as proton donor, 0.1 M of the supporting electrolyte in DMF to yield the cyclized cis and trans products. The reaction with the polymer supported catalyst proceeded with >95% product conversion after 2 hours and yielded a cis:trans ratio of 38:62. These results showed a vast improvement over the non-polymer supported catalyst mediated EHC reaction which resulted in the same cis to trans ratio, but >90% product conversion after 8 hours.
Acknowledgments
We thank Sacramento State Associated Students Inc, IRA grant for a student stipend to Brittanie Clendenin.
Poster #: 286
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Kinase Inhibitition, Drug Discovery, Atropisomerism
Project Title: Structural Optimization of Atropisomeric Pyrrolopyrimidine RET Kinase Inhibitors
Author List:
Toenjes, Sean; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Nalbandian, Chris; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Abstract: Aberrant kinase activity is involved in many different diseases, focusing research efforts towards the development of small molecule kinase inhibitors. Although each kinase plays a specific role in these pathways, the active sites of these kinases are highly conserved throughout the known kinome, making it difficult to selectively inhibit a specific kinase. Inhibitors often bind to both the target kinase and off-target kinases leading to unwanted off-target effects.
Many kinase inhibitors contain at least one rotational axis between two aromatic rings. This leads to an extended form of chirality called atropisomerism, where the two different rotational conformers can either exist as a rapidly racemizing mixture or isolable enantiomers. Most bioactives, as designed, exist as a rapidly interconverting atropisomeric mixture, however, when they bind to target active site, they tend to do so in an atropisomeric fashion. The presence of the non-relevant atropisomer via interconverting atropisomerism or stable racemic atropisomerism can result in off-target inhibition.
In efforts to solve this problem, our lab exploited atropisomerism as a selectivity filter to represent a general strategy to increase kinase selectivity. In our report, we rigidified the biaryl axis by adding steric bulk adjacent to the axis of chirality and found the (R)-conformer to be 5x more selective towards RET kinase than the (S)-conformer after screening across a panel of kinases. The current compounds synthesized in this lab are great early stage ‘lead molecules’ but need to be further optimized for potency and selectivity in order to fully exploit this strategy. To accomplish we first identified potential analogs by screening various substituent combinations of the (R)-configuration using MOE, a molecular modeling software. After conducting QSAR and ranking the results by increased potency, we established a list of priority molecules to be synthesized. We synthesized lead molecules and separated the stable enantiomers via semi-preparative HPLC to obtain enantiopure compounds. We then screened these analogs against RET, YES, SRC, and VEGFR kinase inhibition assays to test for potency and selectivity, consistently realizing atropisomerism was a major contributor towards the selectivity of the tested inhibitors. As more compounds are tested, we will iteratively optimize the computational models to help drive optimization towards RET, potentially leading to valuable chemical probes.
Poster #: 287
Campus: Sacramento State University
Poster Category: Synthetic Chemistry
Keywords: glycodendrimer, carbohydrate, organic synthesis
Project Title: Synthesis of a Hexavalent Cellobiose-terminated Glycodendrimer as an Anti-HIV agent
Author List:
Gwarada, Lucia; Undergraduate, Department of Chemistry, California State University, Sacramento, Presenting Author
McReynolds, Katherine; Department of Chemistry, California State University, Sacramento
Abstract: According to the Foundation of AIDS Research, approximately 36.9 million people, including 2.6 million children are living with HIV/AIDS globally. Currently approved HIV treatments are limited by viral resistance and many side effects. Most of these drugs target the virus after infection. Our research seeks to prevent infection by developing inhibitors that block entry into cells. Dendrimers are a target of our research because they have several sites through which attachment to another molecule can occur (multivalency). Synthesis of dendrimers begins with a core to which linkers can be added to increase the number of ends. Functional groups can be appended to these ends to allow for diverse chemical reactivity. When sugars are added to the ends of dendrimers, glycodendrimers are formed. The sugars can then be sulfated to mimic a host cell’s natural cell surface receptors. These sulfated glycodendrimers have been found to have activity against HIV-1.
In this study, a seven step reaction was used to synthesize a hexavalent glycodendrimer, terminating with cellobiose. Synthesis of the required linker began with a Michael addition using ethylene glycol and acrylonitrile (85% yield). This was followed by a one pot, two step reduction of the nitrile group and subsequent protection of the primary amine with tert-butoxycarbonyl (BOC) group (62% yield). Next, a phthalimide group was added to the linker through a Mitsunobu reaction (42% yield), followed by hydrazynolysis to remove the phthalimide group (77% yield). Next, an oxime-linked cellobiose linker was made by appending cellobiose to the linker (98% yield). This was followed by removal of the BOC group using TFA and dichloromethane (97% yield).
Concurrently, a hexavalent carboxyl terminated core was synthesized. Synthesis began with tris 2-(aminoethyl)amine and a BOC protected, tosylated linker to create a hexavalent BOC protected core. Next, the BOC group was removed yielding a hexavalent carboxyl terminated core, in a two step yield of 65%. In the final step, the core and linker were coupled together to yield the desired cellobiose-terminated glycodendrimer (31% yield). Further research will focus on sulfation of these surface groups, then assays for anti-HIV activity will be conducted.
Funding: Jack Kent Cooke Foundation, NIH ISC3GM119521-01
Poster #: 288
Campus: CSU San Marcos
Poster Category: Synthetic Chemistry
Keywords: Gold(I) catalysis, Ritter Reaction, Microwave
Project Title: The Microwave Assisted, Gold(I)-Catalyzed Ritter Reaction
Author List:
Acosta, Sam ; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author
Ghasb, Elie; Undergraduate, Chemistry & BIochemistry, California State University San Marcos
Iafe, Robert; Chemistry & BIochemsitry, California State University San Marcos
Abstract: The Ritter reaction has become a prominent method for the rapid transformation of alcohols to amides as a result of its atom economy and synthetic ability. The Ritter reaction of nitriles with different compounds constitutes a simple, convenient, one-step methodology to introduce an acylamino function. Despite its synthetic ability, there are several large drawbacks associated with the classical Ritter reaction, which include the use stoichiometric amounts of strong acids, usually sulfuric acid and glacial acetic acid. This requirement limits its application to compounds with acid-tolerant functional groups. As an alternative solution, our lab has developed and optimized the gold(I)-catalyzed Ritter reaction of benzylic alcohols with acetonitrile as the nucleophile using microwave conditions. Despite gold(I)’s traditional characterization as a carbophilic, soft Lewis acid, our chemistry supports gold(I)’s new hard Lewis acid reactivity pattern, where gold reacts similar to the hydronium ion. Although previously thought to be prohibitively expensive and chemically inert, gold actually has many synthetic advantages. For example, gold is relatively difficult to be oxidized, and as a result, many gold-catalyzed reactions can be run without precautions to air and moisture. Surprisingly, gold salts are relatively inexpensive compared to their palladium, platinum, and rhodium counterparts. Multiple different benzylic alcohols were investigated utilizing this new reactivity. The long-term objective is the synthesis of bioactive natural products, unnatural amino acids, and other various building blocks useful in the synthesis of bioactive molecules.
Results: Several different gold(I) and silver(I) salts were used to optimize the Ritter reaction between benzylic alcohols with acetonitrile. It was found that the electron poor tris(perfluorophenyl)phospane ligand on gold afforded the highest yields (quantitative) with benzhydrol, a doubly benzylic alcohol. However, both indanol (77% yield) and phenylethanol (37% yield) reacted best with a N-heterocyclic carbene ligand on gold. Optimal reaction conditions consisted of 5 mol % of both gold and silver salts reacting for 12 hours at elevated temperatures in a microwave reactor. Currently our lab is continuing the investigation of substrate diversity for this gold(I)-catalyzed Ritter reaction and its application to the synthesis of bioactive molecules.
Poster #: 289
Campus: San José State University
Poster Category: Synthetic Chemistry
Keywords: Photosensitizer, light-driven , biocatalysis
Project Title: Fine-tuning the Photosensitizer Properties to Improve the Light-driven Activity of Hybrid P450 Biocatalysts
Author List:
Shalan, Hadil; Undergraduate, Chemistry, San José State University, Presenting Author
Colbert, Alexander; Undergraduate, Chemistry, San José State University
Kato, Mallory; Staff, Chemistry, San José State University
Cheruzel, Lionel; Chemistry, San José State University
Abstract: Biocatalysis involves repurposing enzymes to catalyze chemical reactions, and has recently emerged as an attractive synthetic strategy due to the high specificity of the products formed in contrast to traditional methods. A target of interest for biotechnological applications is the superfamily of P450 heme-thiolate enzymes that functionalize unactivated C-H bonds in a wide range of substrates. Previously, our laboratory has successfully engineered an efficient visible light powered P450 biocatalyst by coupling a bacterial P450 enzyme with a photosensitizing Ru(II)-polypyridyl complex.
The wealth of knowledge on Ru(II)-diimine complexes and their unique photochemistry prompted us to tune their properties in order to further improve the photocatalytic activity of the hybrid enzymes. We systematically modified the hybrid P450 BM3 enzymes with the photosensitizing [Ru(4,4′-R2-bpy)2(PhenA)]2+ (PhenA = [5-acetamido-1,10-phenanthroline; 4,4′-R2-bpy = 4,4′-R2-2,2′-bipyridine). The R substituents included the electron donating (R = N(CH3)2, OCH3, OPh, tBu, CH3, H) and electron withdrawing (R=Cl) groups. A thorough study of analog model complexes was conducted to examine the substituent effects on the photosensitizer properties. The eight model complexes were fully characterized by 1H NMR, mass spectrometry, UV-vis and luminescence spectroscopy as well as cyclic voltammetry to determine their oxidative and reductive potentials. The introduction of the substituents influenced the photochemical properties of the Ru(II)-photosensitizer in terms of light absorption, emission and redox potentials. The hybrid enzymes containing the eight complexes covalently were generated and fully characterized using previously reported techniques. Their photocatalytic activity was readily assessed through the use of a recently reported colorimetric assay compatible with the light-driven reaction conditions. Interestingly, a three-fold increase in turnover rate and number was noticed across the series and could be correlated to the photochemical properties of the photosensitizer. In addition to the characterization of the model complexes and the hybrid enzymes, trends in the photochemical properties of the photosensitizer will be discussed with respect to the optimization of the light-driven process toward biotechnological applications.