2019 CSU Biotechnology Symposium
Posters with Author Listings and Abstracts
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Poster #: 1
Campus: Cal Poly Pomona
Poster Category: Agriculture/Biofuels/Environment
Keywords: Photobiomodulation, Canine, Low Level Laser Therapy
Project Title: Therapeutic Effect of Photobiomodulation Therapy in Canines Following Dental Prophylaxis
Author List:
Watson, Andrea; Graduate, Animal and Veterinary Sciences, California State Polytechnic University, Pomona, Presenting Author
Abstract: Photobiomodulation, or low-level laser, therapy has been documented as a mitigator of wound inflammation and erythema in human and veterinary patients. To determine the therapeutic effect of photobiomodulation therapy in pet canines (Canis lupus familiaris) after dental prophylaxis a low energy GaAlInP laser with a continuous 650 nm wavelength and 100 mW power output was used. Canine owners had the option to enroll their animal (n = 24) in the study if they were receiving an anesthetized dental treatment. During the post-operative recovery, each canine patient received irradiation (10 J/cm2) as a member of one of three randomly assigned treatment groups. One group (n = 7) received four treatment points of irradiation on the gingiva of the right upper and lower dental arcade, one group (n = 8) received four treatment points of irradiation on the gingiva of the left upper and lower dental arcade, and one group (n = 9) received a mock gingiva treatment without irradiation. Inflammation and erythema along the gingival treatment area of each canine was scored 24 hours after treatment by a blinded veterinary evaluator in person or via photograph. There was no significant difference between the gums of the canines in the right side treated group and the canines in the control group (P = 0.20) when the upper right dental arcade was evaluated. There was also no significant difference between the gums of the canines in the left side treated group and the canines in the control group (P = 0.14) when the upper left dental arcade was evaluated. The canine patients receiving treatment did show an overall clinical decrease in inflammation and erythema, but this was not statistically significant due to a limited sample size.
Poster #: 2
Campus: Cal Poly San Luis Obispo
Poster Category: Agriculture/Biofuels/Environment
Keywords: Sub-zero saline chilling, quality improvement , Safety imrprovement
Project Title: Improvement of Chilling Efficiency, Meat Quality, and Microbial Safety of Broiler Carcasses Using Sub-zero Saline Solution Chilling
Author List:
Viliani, Samira; Graduate, Animal Science, California Polytechnic State University, San Luis Obispo, Presenting Author, Eden Award Nominee
Morgan , Metheny; Staff, Animal Science , California Polytechnic State University, San Luis Obispo
Lee, Hongchul; Postdoc, Animal Science , California Polytechnic State University, San Luis Obispo
Bennett, Darin ; Faculty, Animal Science , California Polytechnic State University, San Luis Obispo
Hurley , Sean ; Faculty, Agribusiness, California Polytechnic State University, San Luis Obispo
Kang , Ike ; Faculty, Animal Science , California Polytechnic State University, San Luis Obispo
Abstract: The purpose of this research was to investigate the effect of sub-zero saline chilling on chilling efficiency, product quality, and microbial safety of broiler carcasses. In Experiment I, II, and III, eviscerated broiler carcasses were chilled using high salt (4% NaCl/-2.41oC, and 8% NaCl/-5.08oC), low salt (1% NaCl/-0.6°C, 2% NaCl/-1.2°C, and 3% NaCl/-1.8°C), and intermediate salt (3% NaCl/-1.8°C, and 4% NaCl/-2.41oC) solutions, respectively. An ice slurry control (0% NaCl/0.5oC) was used for all experiments. Data in three replications were evaluated by one-way ANOVA and a completely randomized design. A post-hoc analysis was performed using Duncan`s multiple range test to evaluate difference among treatments at P < 0.05. The chilling time of broiler carcasses in subzero saline solutions was reduced by 11% in 3% NaCl/-1.8 oC and 37% in 4% NaCl/-2.41 oC over the ice slurry control. The breast tenderness of broiler carcasses in 8% NaCl/-5.08 oC were significantly improved than those in 3% NaCl/-1.8 oC (P < 0.05), with an intermediate value observed in 4% NaCl/-2.41 oC. The bacteria populations of mesophilic aerobic bacteria (MAB), Escherichia coli (E. coli), and total coliforms were significantly reduced when carcasses were chilled in 3% NaCl/-1.8 °C and 4% NaCl/-2.41 °C than the water solution (P < 0.05), except the MAB. Considering the salt concentration to reduce in chilling solutions and meat tenderness to improve in broiler carcasses, we concluded that carcass chilling in 4% NaCl/-2.41 °C solution appears to be the right choice. Additional research is required to investigate product sensory analysis, lipid oxidation of carcasses, and overall processing costs.
Poster #: 3
Campus: CSU Long Beach
Poster Category: Agriculture/Biofuels/Environment
Keywords:
Project Title: Insights into the Structure and Function of the Gut Microbiome in Cartilaginous Fishes
Author List:
Freund, Hannah; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Winans, Natalie; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Martinez-Steele, Laura; Undergraduate, Biological Sciences, California State University, Long Beach
Lowe, Chris; Undergraduate, Biological Sciences, California State University, Long Beach
Berlemont, Renaud; Faculty, Biological Sciences, California State University, Long Beach
Abstract: Vertebrates depend on their gut microbiome to produce most of the digestive enzymes such as glycoside hydrolases (GHs) involved in the processing of polysaccharides. Consequently, microbes within the gut can affect the physiology of the host, its ecological niche, and its trophic habits. Conversely, microbes are affected by the ontogenetic changes of their host. However, excluding the human gut, there is little to no information about how the host’s development affects the associated gut microbiome. Thus, here we examined the structure and function of the microbial community in the spiral valve intestine of two sympatric cartilaginous fishes (i.e. Heterodontus francisci and Pseudobatos productus). More precisely, we investigated how changes in the fishes’ ecological niche, as they mature, affects the microbiome in their gut using next-generation sequencing (NGS) and bioinformatics.
Both the taxonomic and functional diversity within the sequenced metagenomes were identified using the MG-RAST annotation platform and custom bioinformatics approaches. Eventually we identified a significant increase in the frequency of genes coding enzymes targeting chitin (the major constituent of the shellfish exoskeleton) in mature P. productus only. We thus hypothesized that this reflected variation in the diet of these fish. In order to test this, we developed an original bioinformatics pipeline based on eukaryotic COX-genes annotation to identify the origin of the “food-DNA” in the digesta. This approach allowed us to further investigate the relationship between the structure and function of the gut microbial communities, and the development and feeding habit of the fishes.
Poster #: 4
Campus: CSU Northridge
Poster Category: Agriculture/Biofuels/Environment
Keywords: biodiesel, plant biotechnology, Arabidopsis thaliana
Project Title: Synthesis and evaluation of germacrene, a potential biodiesel compound, in Arabidopsis thaliana
Author List:
Cerda, Christopher; Undergraduate, Biology, California State University, Northridge, Presenting Author
Cox-Georgian , Destinney
Basu, Chhandak; Faculty, Biology, California State University, Northridge, Presenting Author
Abstract: The global energy crisis is dependent on fuel sources that are becoming more scarce, which makes the search for alternative renewable energy resources vital. Sources of fossil fuels such as coal and oil are non-renewable, environmentally harmful, and are depleting quickly. A solution to the energy crisis is to create a renewable source of biodiesel using terpene molecules generated in the seed oil of plants. A subcategory of terpenes called sesquiterpenes have similar molecular structures compared to diesel fuel compounds (C15H24) and can be used as a fuel source. In this project, we performed Agrobacterium-mediated genetic transformation using two Arabidopsis thaliana plants overexpressing a germacrene synthase gene that is responsible for producing the germacrene C sesquiterpene. The germacrene synthase gene was cloned into a recombinant plasmid vector under the control of a constitutive promoter and kanamycin resistance gene as a selectable marker. An Agrobacterium strain containing the plasmid vector was used to transform Arabidopsis thaliana plants using the ‘floral dip’ method. Transformants were identified using antibiotic selection, and integration was confirmed by amplifying the NPT II resistance gene using PCR and gel electrophoresis. The oil content of seeds will be evaluated by GCMS, and qPCR will be used to determine the quantification of gene expression compared to wild-type controls. Expression of germacrene synthase gene in Arabidopsis thaliana was a proof of concept. We are in the process of transforming tobacco and sesame plants with germacrene synthase genes. Commercial biodiesel is produced by transesterification of oils, so overproduction of sesquiterpenes in sesame seed oil should improve overall quality and yield of biodiesel produced from the transgenic plants. Successful execution of the project in tobacco and sesame will demonstrate the feasibility of this procedure for biodiesel production in plants which can provide a renewable source of fuel.
Poster #: 5
Campus: CSU San Marcos
Poster Category: Agriculture/Biofuels/Environment
Keywords: Arabidopsis thaliana, glutaredoxin, development
Project Title: The Arabidopsis thaliana glutaredoxin AtGRX660 controls lateral root development and shoot organ size
Author List:
Carpinelli, Sophia; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Cowling, Craig; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Rosas, Miguel; Staff, Biological Sciences, California State University San Marcos
Escobar, Matthew; Faculty, Biological Sciences, California State University San Marcos
Abstract: Glutaredoxins (GRXs) are small oxidoreductase enzymes that can reduce disulfide bonds in target proteins. The genome of the model plant Arabidopsis thaliana has more than 30 GRX genes, but the biological function of most of these GRXs is unknown. We previously found that a small group of Arabidopsis GRX genes is specifically activated by nitrate, a common source of nitrogen in the soil. In order to better characterize the function of one of these nitrate-regulated GRXs, AtGRX660, we generated transgenic Arabidopsis plants that continuously overexpress the AtGRX660 gene. We isolated RNA from 12 independent transgenic lines and quantified AtGRX660 mRNA levels via real-time reverse transcriptase PCR. Three elite lines displaying >100-fold increase in basal AtGRX660 transcript levels were selected for further analysis. AtGRX660-overexpression lines and wild-type plants were grown on soil in a controlled environment growth chamber for characterization of shoot phenotypes, and on vertically-orientated plates of plant growth media for characterization of root phenotypes. All AtGRX660-overexpression lines displayed a dwarf shoot phenotype, with significant reductions in shoot biomass, total leaf area, and silique length compared to wild-type plants. In addition, root system architecture was highly altered. While primary root growth was normal in the transgenic plant lines, lateral roots were almost completely absent. Phase contrast microscopy demonstrated that lateral root primordia develop in the transgenic lines, but these primordia do not elongate and emerge from the primary root. Overall, these results suggest that AtGRX660 acts a negative regulator of shoot organ development and inhibits lateral root elongation. Our findings could have agricultural relevance in plant drought tolerance, since AtGRX660 differentially affects primary root system growth (root system depth) and lateral root system growth (root system breadth).
Research supported by NSF IOS grant 1651584 (ME), Trio McNair Scholars Program (CC), and CSUPERB Presidents’ Scholars Program (SC).
Poster #: 6
Campus: San Diego State University
Poster Category: Agriculture/Biofuels/Environment
Keywords: Arabidopsis, drought stress, salt stress
Project Title: Comparative analysis of Arabidopsis and Boechera abiotic stress tolerance.
Author List:
Epstein, Ruth ; Undergraduate, Biology, San Diego State University, Presenting Author
Leoni, Kaeli; Undergraduate, Biology, San Diego State University, Presenting Author
Waters, Elizabeth; Faculty, Biology, San Diego State University
Abstract: Rising levels of carbon dioxide in the atmosphere, caused by the burning of fossil fuels, has altered the earth’s climate. Global climate change has increased overall temperatures and increased the frequencies of extreme temperature events such as heat waves and drought. At the same time that this is occurring the human population has continued to increase. This has put strains on agriculture. More and more farmers are using irrigation water to keep crops alive and irrigation increases the amount of salt in the soil. Many agricultural regions have experienced decreases in productivity due to drought (water scarcity) and salt contamination of soils. There is a great need to develop crop species that can tolerant both drought and salt stress. We have been studying the Boechera genus (Brassicaceae) to help identify novel pathways of plant stress tolerance. The Boechera genus is found only in Western North American and a number of Boechera species are native to California. Previously we have shown that a population of Boechera depauperata from a high mountain ridge in the Sierra Nevada is highly tolerant to high temperature stress. Here we describe the tolerance of B. depauperata to both drought and salt stress. We have compared the levels of tolerance of B. depauperata to that of Arabidopsis thaliana (a model plant). We have grown B. depauperata for two weeks and then withheld water. At two-day intervals we have sampled stressed and non-stressed plants and determined the extent of cell death in leaves and the activity of Photosystem II (PSII) (a crucial component of photosynthesis). In addition, we have exposed two-week old plants to 50 mM NaCl, 100 mM NaCl, and 200 mM NaCl. At two-day intervals we again measured cell death and PSII activity. This data shown that while B. depauperata is more tolerant than A. thaliana to heat stress, it is less tolerant to salt stress. This suggests that the pathways that provide heat stress in B. depauperata are not overlapping with those that provide salt stress tolerance.
Poster #: 7
Campus: CSU Bakersfield
Poster Category: Agriculture/Biofuels/Environment
Keywords: Vitus vinifera l., deep learning, pierces disease
Project Title: A Mobile Application to Detect Grapevine Pierce’s Disease with Neural Networks
Author List:
Smizer-Muldoon, Kyle; Undergraduate, Dept. Computer and Electrical Engineering and Computer Science, California State University, Bakersfield, Presenting Author
Osagie-Amayo, Obosa; Undergraduate, Dept. Computer and Electrical Engineering and Computer Science, California State University, Bakersfield, Presenting Author
Cruz, Alberto; Faculty, Dept. Computer and Electrical Engineering and Computer Science, California State University, Bakersfield
El-Kereamy, Ashraf; University of California Cooperative Extension Kern County
Ampatzidis, Yiannis; Dept. Agricultural and Biological Engineering, University of Florida
Abstract: Xylella fastidiosa is the pathogen responsible for Pierce’s Disease in Vitus vinifera L. In 2014, PD caused $104.4 million losses to Californian wine and table grape growers. Infections remain a problem despite efforts to control the glassy winged sharpshooter, the primary vector. It continues to be a problem in Napa and Sonoma where control strategies are ineffective due to climate and soil. In conventional testing, petioles are collected after the leaf blades have dried. Delays in detection risk contamination of neighboring vines. However, infected vines display leaf scorching which can be identified three to eighteen months after initial infection.
We propose a system to automatically detect PD from leaf clipping photos taken with a mobile device. The system provides early screening to improve reaction time of control strategies. It can be deployed earlier than lab tests and at a lower cost. The program does not require the user to be an expert at identifying the PD and it is fully automatic.
PD symptoms are easily confused with other diseases and conditions. For effective detection, we use deep learning to detect leaf scorch cues indicating the presence of PD. We are the first to apply deep learning to detection of grapevine PD. Leaf clippings of six grape varieties were photographed with an RGB camera and infection was verified by commercial laboratory analysis. Data was augmented from the publicly available PlantVillage dataset. Populations include control, PD, black rot, esca, leaf spot and a category of non-grapes and range from 600 to 1300 samples. We employ a pre-trained, fine-tuned AlexNet deep learning architecture and achieve an accuracy of 97.18%. For reference, color features and a linear support vector machine have an accuracy of 85.70%. The deep learning algorithm was programmed in Python with Theano. The mobile app consists of four parts: the database, using PostgreSQL; REST API, built with Python to interface with Theano; a front-end app to report analytics built in React; and a user GUI built with React Native and Redux. Future work will focus on collecting samples of abiotic stress which are often a false positive for PD.
Acknowledgement: CSUPERB NI 2018, CSUB PDP Mini-Grant 2018
Poster #: 8
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Biochemistry, Biophysics, Lipoproteins
Project Title: Biochemical and Biophysical Analysis of Apolipoprotein (apo)E3/apoE4 heteromerization
Author List:
Abhari, Devan; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Tu , Kai-Han ; Graduate, Chemistry and Biochemistry, California State University, Long Beach
Narayanaswami, Vasanthy; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract: Apolipoprotein E (apoE) is a 299 residue, exchangeable apolipoprotein that has essential roles in plasma cholesterol homeostasis and reverse cholesterol transport. It has the ability to exist in lipid-free and lipoprotein-associated states. ApoE is a two-domain protein with the C-terminal (CT) domain mediating protein self-association to form dimers and tetramers via helix-helix interactions. In humans, the APOE gene is polymorphic with 3 common alleles, ε2 ε3 and ε4, occurring in frequencies of 8, 77 and 15%, respectively in the population. Heterozygotes expressing both apoE3 (Cys112) and apoE4 (Arg112) isoforms represent the highest population of ε4 carriers, an allele highly associated with Alzheimer’s disease. In this study we test the hypothesis that apoE3 and apoE4 hybridize to form a heteromer in lipid-free state. We employed a combination of biochemical and biophysical approaches to test the hypothesis using recombinant purified His-tagged or FLAG-tagged apoE3 and apoE4. Refolding an equimolar mixture of His-tag/apoE3 and FLAG-tag/apoE4 (or vice versa) followed by capture with anti-FLAG antibody and immunoblotting with anti-His-tag antibody (or vice versa, capture with cobalt-conjugated Dynabeads and immunoblot with anti-FLAG antibody) indicated association of His- and FLAG-tagged proteins. Forster resonance energy transfer between donor fluorophore on one isoform and acceptor on the other, both located in the respective CT domains, revealed a distance of separation of ~ 40 Å between the donor/acceptor pair indicative of spatial proximity between the CT domains of apoE3 and apoE4. Similarly, a quencher placed on one was able to mediate significant quenching of fluorescence emission on the other, indicative of spatial proximity within collisional distance between the two. Taken together, the data indicate that apoE3 and apoE4 can undergo protein-protein interaction to form heteromers. The possibility of heteromerization of apoE3/apoE4 bears implications in the physiological behavior of these isoforms in terms of lipoprotein binding and conferring atheroprotection or increasing the risk for Alzheimer’s disease.
This work is supported by a grant from the National Institutes of Health award # GM105561
Poster #: 9
Campus: Sonoma State University
Poster Category: Biochemistry
Keywords: persulfides , chemical biology ,
Project Title: Synthesis and Characterization of Cobalt (II/III) Salen Complexes and their Interactions with Persulfide Species
Author List:
Holm, Annika ; Undergraduate, Chemistry , Sonoma State University, Presenting Author
Fukuto , Jon ; Faculty, Chemistry , Sonoma State University
Works, Carmen ; Faculty, Chemistry , Sonoma State University
Abstract: Persulfide species (RSSH) have been of particular interest due to their unique role in biological processes. Among all biologically relevant sulfur species, persulfides are likely the most reducing. It is proposed that persulfides will readily reduce oxidized metalloproteins. Specifically, persulfides have the potential to take Fe(III) myoglobin and reduce it to the oxygen carrying form. The goal of this research project is to identify interactions between biologically relevant metal complexes and persulfides. Co(II) and Co(III) Salen Cobalt complexes were synthesized and characterized, and their reactivity with persulfide donors was studied using optical spectroscopy and mass spectrometry. Methoxycarbonyl persulfide donor (MCPD) was used to test for possible metal-persulfide or ligand-persulfide interactions. Through an intramolecular rearrangement, the persulfide is unmasked and reacts generally as a strong nucleophile in the presence of electrophilic molecules. The donor was reacted with both Co(II) and Co(III) Salen complexes to further explore reactivity of persulfides with a range of biologically important transition metals . Cobalt Salen was selected because both Co(II) and Co(III) are stable states, and its behaviors parallel other metalloprotiens (reversible binding of O2). Differences in UV-Vis spectra between Cobalt (II) and (III) allow us to determine if electron transfer is occurring, and other analytical tools like mass spec and IR help to determine what the products of this electron transfer are. Results showed that there was an interaction between Co(II) and the persulfide donor, as spectral changes indicated immediate oxidation of the cobalt. A secondary reaction is also observed once the persulfide has been unmasked from the donor molecule. Results indicate that some chemistry may be occurring between the persulfide species and the metal complex, either through direct interaction with the metal or through metal assisted ligand modification. There were no spectral changes observed when Co(III) was reacted with the persulfide. This could be due to the Co(III) Salen synthesis method possibly inhibiting a necessary active site of the molecule, or due to the presence of a stronger oxidizing agent. Current work is focused on identifying the process and the nature of the initial reaction between donor and Co(II) complex, and also the participants in the secondary reaction once the persulfide specie has been generated.
Poster #: 10
Campus: CSU Los Angeles
Poster Category: Biochemistry
Keywords: breast cancer, drug resistance, Her2
Project Title: The effect of t-Darpp Thr-39 phosphorylation on Protein Kinase B (AKT) activation in Herceptin-resistant breast cancer
Author List:
Singh, Sarwyn; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Avanes, Arabo; Staff, Chemistry and Biochemistry, California State University, Los Angeles
Momand, Jamil; Faculty, Chemistry and Biochemistry, California State University, Los Angeles
Abstract: Her2-type breast cancers are characterized by overexpression of Her2 receptor protein, typically due to increased ERBB2 gene copy number. These fast-growing cancers account for 17-25% of breast cancers in the US, resulting in high levels of mortality. The clinically-approved antibody drug trastuzumab (Herceptin) binds to Her2 and inhibits its signaling activity (Her2 -> PI3 kinase ->AKT). However, within an average of one year, Her2-type cancers resurge due to acquired resistance resulting in AKT reactivation (AKT phosphorylation at S473). When breast cancers acquire resistance, they frequently overexpress t-Darpp. Forced expression of t-Darpp in Herceptin-sensitive breast cancer cells induces partial Herceptin-resistance. In such cells, t-Darpp also increases protein kinase A (PKA) activity and AKT phosphorylation, both important contributors to cell survival and proliferation pathways. How t-Darpp activates PKA and AKT remains unknown. Previously, it has been suggested that phosphorylation of T39 on t-Darpp correlates with PKA activity and AKT phosphorylation. To rigorously test the effect of T39 phosphorylation on AKT phosphorylation we used isogenic SKBR3 cells expressing wild-type t-Darpp, t-Darpp with an alanine mutation at T39, and t-Darpp with an aspartate mutation at T39 (mimicking the phosphorylated state) for effects on AKT. We found that all three cell lines exhibit two-fold increased AKT phosphorylation compared to empty vector-transfected control cells. There was no effect on AKT protein levels. This indicates that a region of t-Darpp outside of T39 is responsible for mediating AKT phosphorylation. To explore whether PKA activation is necessary for AKT phosphorylation, PKA will be knocked down with siRNA directed against PKA catalytic subunit alpha (PKA-ca) and relative AKT phosphorylation level will be measured.
Poster #: 11
Campus: CSU Los Angeles
Poster Category: Biochemistry
Keywords: Drosophila, BMP, Phosphatase
Project Title: A screen to identify new modulators of the Bone Morphogenetic Protein signaling pathway
Author List:
Castro, Kenny; Undergraduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Eiley, Courtney; Undergraduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Eivers, Edward; Faculty, Biological Sciences, California State University, Los Angeles
Abstract: Bone morphogenetic proteins (BMPs) are a family of cytokines that are involved in a multitude of biological processes, from determining tissue types in the early embryo, to maintaining tissue homeostasis in the adult organism. Even with our growing knowledge of this signaling pathway during development and disease, there are still many unanswered questions that continue to drive research in this field of signaling. Broadly, our projects goal was to identify new phosphatases which modulate the BMP pathway during Drosophila wing development. Phosphatases operate by dephosphorylating phosphorylated proteins which typically results in activation or inactivation of a protein of interest. This experimental goal was achieved by overexpressing inducible RNAi’s in developing fruit fly first instar wing imaginal discs and analyzing resultant adult wing phenotypes. In total we screened 70 different serine/threonine protein phosphatases and analyzed adult Drosophila wings for typical BMP gain-of function or loss of function phenotypes (examples: gain or loss of wing vein tissue). Phenotypic analysis of potential positive hits using light microscopy included incomplete crossveins, ectopic longitudinal vein tissue, loss of longitudinal vein five and complete loss of adult wings. To date we have identified and are pursuing ten new potential BMP pathway modulators. Currently, we are investigating these positive hits further by analyzing the phosphorylation state of the BMP pathway transcription factor Mad using Western blotting and cultured cell immunostaining. In conclusion, our experiments aim to expand our knowledge of the BMP pathway by identifying new pathway modulators, which will advance our understanding of the basic molecular mechanisms which act to control the BMP signal at the level of Mad phosphorylation-dephosphorylation.
This work was supported by NIH grant R15GM123374
Poster #: 12
Campus: CSU San Bernardino
Poster Category: Biochemistry
Keywords: influenza virus , protein interactions , gradient sedimentation
Project Title: Characterizing NP mutant interactions to reveal novel antiviral targets
Author List:
Atkins , Cody J. ; Undergraduate, Biology , California State University, San Bernardino, Presenting Author
Gallardo, Jennifer ; Undergraduate, Biology , California State University, San Bernardino, Presenting Author
Newcomb , Laura ; Faculty, Biology , California State University, San Bernardino
Abstract: Influenza A viruses cause widespread outbreaks of respiratory infections that are life-threatening and fatal in some cases. The machinery that is responsible for influenza RNA synthesis is the viral ribonucleoprotein (vRNP), comprised of multiple nucleoproteins (NP), the RNA genome segment, and the RNA dependent RNA polymerase (RdRP), a trimer consisting of PA, PB1, and PB2. NP interactions are targets for novel influenza inhibitors because NP is highly conserved among influenza subtypes and an essential component of functional vRNPs. NP interacts with viral factors such as two subunits of RdRP, PB2 and PB1, and host factors such as UAP56, a DEAD box RNA helicase. Here, we use NP mutants shown to be defective in vRNP function to investigate NP interactions, with the goal to identify novel antiviral targets. Specifically we examined two NP mutants: NPbd3, a NP mutant with 5 amino acid glycine substitutions within the body domain of NP hypothesized to disrupt the NP-PB2 interaction, and del20NP, a NP mutant with the N-terminal 20 amino acids deleted reported to disrupt interaction with UAP56. Human tissue culture 293T cells were transfected with wild type NP or NP mutant alone and in combination with PB2. Cells were collected 48 hours post transfection and total protein was isolated. Protein extracts were separated on a 5-40% sucrose gradient. After ultracentrifugation for 16-hours at 50k rpm and 4°C, fractions were collected and each fraction was analyzed by Western blot. Multiple trials confirm wild type NP co-sediments with PB2 while NPbd3 does not. This data supports our hypothesis that NPbd3 is non-functional for vRNP activities because its interaction with PB2 is disrupted. We aim to examine double mutants within this NP body domain to characterize NP-PB2 interaction further. NP-UAP56 interaction analysis proved less conclusive. Wild type NP did not show clear co-sedimentation with UAP56, consistent with previous co-immunopurification of NP-UAP56 which could not confirm the reported NP-UAP56 interaction. However, in the presence of PB2, WT-NP did co-sediment with UAP56. The fact we observed interaction in the presence of PB2 is intriguing and we have begun sedimentation analysis with addition of PB2 to cells expressing del20NP or NPbd3 mutant. Our research will characterize NP interactions which may provide multiple targets for novel antivirals that can be used in combination to combat Influenza.
Poster #: 13
Campus: CSU Bakersfield
Poster Category: Biochemistry
Keywords: Hops, Cancer, Phytochemicals
Project Title: The Potential Intrinsic Anticancer Characteristics of Hops
Author List:
Gonzales, Alexander; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield, Presenting Author
Song, Thomas; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield, Presenting Author
Sandoval, Omar; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Rios, Diana; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Kaur, Navdeep; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Singh, Henna; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Arechiga, Daisy; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Davis, Isabel ; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Rotella, Donna; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Forester, Sarah; Faculty, Chemistry and Biochemistry, California State University, Bakersfield
Abstract: Terpenes, alpha acids, and beta acids derived from hops (Humulus lupulus) contribute to the flavor, aroma, bitterness, and preservative characteristics of craft beer. These molecules dissolve during the boil of the wort; however, the alpha acids isomerize while terpenes and beta acids remain unchanged in the solution as it passes on to the final product. Some of these molecules, such as the terpene alpha-humulene, have been shown to inhibit the growth of cancer cells. This study investigates the contribution of these intrinsic bioactive components from hops in the potential anticancer activity of beer. Two beer samples (Hollow Body & El Cerrito) and two solutions containing only the hops (Citra/El-Dorado & Saaz) were used to make the beer before being freeze-dried and resuspended in cell culture media at original brew strength and varying diluted concentrations. HT-29 human colon cancer cells were incubated with various concentrations of the beer and hops treatments for 72 hours. Cell viability was quantified using the thiazolyl blue tetrazolium bromide (MTT) assay. The results indicated that at low concentrations, the beer treatments stimulated proliferation of the cancer cell line by 206.4% (Hollow Body) and 265.0% (El Cerrito). However, as the solutions approached original brew concentration, inhibition of cell proliferation was observed. At all concentrations, the hops solutions inhibited cell proliferation. Specifically, cell growth was inhibited by 33.5% (Citra/El Dorado) and 29.5% (Saaz) at the original brew concentration. To investigate the proliferative effects of the beer treatments, the samples were analyzed for total carbohydrates (phenol-sulfuric acid method), residual sugars (Lane-Eynon method), phenols (Folin Ciocalteu method), antioxidant activity (Oxygen Radical Absorbance Capacity method), and alpha-humulene concentration (gas chromatography – mass spectrometry). The beer samples had greater concentrations of total carbohydrates and residual sugar, and less total phenols, antioxidant activity and alpha-humulene compared to the hops samples. We conclude that the proliferative effects of the beer treatments resulted from high levels of carbohydrates such as simple sugars. Additionally, the anti-proliferative effects of the hops treatments potentially resulted from bioactive molecules such as polyphenols and alpha-humulene. In the future, alpha and beta acids from the hops will be isolated and used to treat cancer cells.
Poster #: 14
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Polypeptides, High-resolution NMR spectroscopy, Caspase 2
Project Title: Structural Diversity by Point Mutation in Heptapeptides: Multi-Dimensional NMR Studies
Author List:
Bentley, Timothy; Graduate, Chemistry, California State University, Fresno, Presenting Author
Hernandez, Tess; Graduate, Chemistry, California State University, Fresno, Presenting Author
Garcia, Raymond; Undergraduate, Chemistry, California State University, Fresno
Krishnan, Krish; Faculty, Chemistry, California State University, Fresno
Maitra, Kalyani; Faculty, Chemistry, California State University, Fresno
Abstract: Structural features of a polypeptide are reliant upon the residues of the amino acid sequence, which in turn, controls its function and activity. Proteins such as Caspase 2 found in mammals and the tRv2048c complex found in Mycobacterium tuberculosis are known to bind to polypeptides containing a specific amino acid sequence alanine, aspartic acid, and valine (ADV). Caspase 2 is inhibited via a capped pentapeptide, while the tRv2048c complex inhibitor has the same overlapping sequences of amino acids. Inspired by the commonality of the protein inhibitors and to further understand the role of amino acid sequence-structure dependence, the peptide ADVADVG, and a derivative was synthesized via automated synthesizer, using solid phase chemistry, and Fluorenylmethyloxycarbonyl (Fmoc) protected amino acids. The three-dimensional structure of the peptides was determined using high-resolution NMR spectroscopy, which utilized peak analysis from total correlation spectroscopy (TOCSY) and rotating-frame Overhauser spectroscopy (ROESY), in tandem with the 3D modeling software. The overall 3D structure of ADVADVG was found to be reliant upon the fourth residue, alanine, which interacts with the aspartic acids and valines side chains. The structure further shows a turn, centered at the fourth alanine residue that is stabilized by several intra-residue hydrogen bonds. It was then hypothesized that the residue at fourth position is critical for the 3D structure of the peptide and that mutating the 4th position, with a more non-polar hydrophobic residue, will increase the side chain interactions between the aspartic acid and valine residues, through intra-residue hydrogen bonds. The derivative, ADVFDVG, was synthesized and characterized with the belief that the phenylalanine would provide an increased hydrophobic environment, due to the hydrophobic-aromatic ring. In support of the hypothesis, the derived polypeptide displayed an increased amount of side chain inter-residue interactions between the alpha and beta protons of the phenylalanine and both valines; however, the side chain lacked affinity to the more polar aspartic acids. In the future, further mutations will be introduced in place of the fourth residue, and computational analyses will be performed to determine potential protein inhibition via docking simulations.
Poster #: 15
Campus: Humboldt State University
Poster Category: Biochemistry
Keywords: bioremendiation, Surface layer,
Project Title: Nanoscale Modifications to the RsaA S-Layer Protein Enhance Lead binding in Whole Cells
Author List:
Templeton, Kayla; Undergraduate, Chemistry, Humboldt State University, Presenting Author
Coblentz, Azariah; Humboldt State University, Chemistry, Humboldt State University
Cappuccio, Jenny; Faculty, 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. C. vibrioides strains were morphologically characterized though transmission and scanning electron microscopy. Engineered strains were genetically verified and found 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 #: 16
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: apolipoprotein, protein stability, salt bridge
Project Title: Lysine 52 contributes to the protein stability of the helix bundle of apolipophorin III
Author List:
Tran, Angela; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Weers, Paul; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract: Apolipophorin III (apoLp-III) is an 18 kDa exchangeable apolipoprotein found in insects. The protein is used as a model to better understand lipid transport processes and innate immunity due to the availability of three-dimensional structures of the protein, allowing a structure-guided approach. ApoLp-III from Locusta migratoria contains eight lysine residues, which can interact with negatively charged phospholipids of bacterial membranes. They also help to maintain the structure of the protein by ionic interactions, as removal leads to a protein devoid of secondary structure. Denaturation studies with guanidine-HCl showed that when lysine residues 52 and 54 were changed into glutamine, protein stability was significantly reduced and indicated disruption of a salt bridge. To identify which of the two lysine residues form a salt bridge with neighboring glutamate or aspartate residues, site-directed mutagenesis was employed to design single lysine to glutamine (KQ) variants. The K52Q and K54Q single variants were generated using the Quick-change site-directed mutagenesis kit, and the correct amino acid change was verified by DNA sequencing. The proteins were expressed in E. coli and purified by size-exclusion chromatography and reversed-phase HPLC. Circular dichroism was employed to determine the secondary structure content and protein stability. Far UV circular dichroism scans showed a modest increase in helical content from 67% in the wild type protein to 73% for K52Q-apoLp-III and 83% in K54Q-apoLp-III. The proteins were denatured with increasing concentrations of guanidine-HCl to determine their resistance to the denaturant. The midpoint of guanidine-induced denaturation, which is used as a measure for protein stability, decreased from 0.50 ± 0.01 M for wild-type apoLp-III to 0.42 ± 0.06 M for K52Q-apoLp-III. The midpoint of denaturation did not change for K54Q-apoLp-III (0.48 ± 0.02 M). The decreased protein stability for K52Q-apoLp-III showed that lysine 52 aids in the stabilization of the protein structure, presumably through an interhelical salt bridge with aspartate 90, which is positioned in close proximity. Thus, while lysine residues are located on the protein surface, they can be contributing to the protein structure and stabilize the 5-helix bundle. Solubilization of anionic and zwitterionic phospholipid vesicles will be used to verify if this amino acid change also affects the lipid binding properties of apoLp-III.
Poster #: 17
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: citrate synthase, larvae, enzyme
Project Title: Using the Aerobic Enzyme, Citrate Synthase, to Understand Biogeographic Dispersal Potential in Echinoid Larvae
Author List:
Pouv, Amara; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Ohanian, Alec; Undergraduate, Biological Sciences, California State University, Long Beach
Pace, Douglas; Faculty, Biological Sciences, California State University, Long Beach
Abstract: Temperature is a primary determinant of biogeographic distribution in animals due to its influence on biochemical processes, especially aerobic metabolism. This study used the aerobic, Kreb’s cycle enzyme, citrate synthase (CS) to determine if: 1) habitat ranges of adult echinoids are linked to temperature sensitivity of critical metabolic enzymes during the larval stage, and 2) in vitro activity of metabolic enzymes can be used to predict physiological state of larvae. The thermal performance of CS was determined in three echinoid larvae with the following thermal habitat ranges: Dendraster excentricus (2-28˚C), Strongylocentrotus purpuratus (2-24˚C), and Strongylocentrotus fragilis (0-10˚C). In vitro analysis of CS activity and temperature sensitivity (from 5-30˚C) was determined using Q10 and Arrhenius break point analysis for larvae of each species. D. excentricus had consistent Q10 values across all temperatures and displayed no discrete change in activation energy. S. fragilis and S. purpuratus displayed diverging Arrhenius breakpoints at a common temperature of 15˚C, where decreasing temperatures resulted in increased activation energy for S. purpuratus but decreased values for S. fragilis; supporting the assertion that despite overlapping temperature ranges echinoderm larvae can possess significant differences in metabolic performance. In furtherance of this observation, the ability to predict in vivo physiological state with in vitro CS activity (Aim 2) was assessed by correlating total CS activity with in vivo, whole larval metabolic rate. Metabolic rates of both S. fragilis and D. excentricus were highly correlated with CS activity (regression analysis yielded p-value < 0.002 and R2 > 0.9 in both species). However, D. excentricus possessed 3-times more CS activity per unit of metabolic rate compared to S. fragilis larvae. Further experiments will determine the CS-metabolic relationship in larvae of S. purpuratus, which is closely related to S. fragilis but is more eurythermal and typically found in shallower, warmer waters. The results of this research demonstrate how thermal dependence of early life-history metabolic pathways have ramifications for dispersal potential and setting physiological state in the planktotrophic larvae of benthic marine organisms.
Poster #: 18
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Biochemistry, Lipoproteins, Endothelial biology
Project Title: Investigating transcytosis of apolipoprotein AI in bovine aortic endothelial cells
Author List:
Meyer, Kyle; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Nguyen, Tina; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Narayanaswami, Vasanthy; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract: Despite the vast amount of literature indicating low plasma levels of high density lipoprotein-cholesterol (HDL-C) as a risk factor for cardiovascular disease (CVD), the last decade has seen a paradigm shift in the concept that functionality of HDL may be more important than its level in the plasma as a determining factor in CVD. The inverse correlation between plasma HDL-C levels and CVD risk has been questioned by numerous studies showing evidence that neither pharmacological nor genetic intervention to increase HDL-C levels lowered the risk for CVD. Thus, there is a need to understand the role of HDL in CVD from a mechanistic perspective. HDL are large lipid-protein complexes with apolipoprotein (apo) AI being a major component and an important player in cholesterol transport. The goal of this project is to understand HDL transcytosis as it traverses the endothelial layer lining the arterial intima and to investigate possible structural and functional alterations to the HDL as a consequence of this transcytosis. We tested the hypothesis that apoAI undergoes transcytosis in either the lipid-free or lipid-associated state. To answer this, bovine aortic endothelial cells (BAOEC) were grown to confluence on Transwell inserts, establishing tight junctions. Recombinant apoAI with a hexa-His tag was over-expressed in E. coli and purified by affinity and size exclusion chromatography. The protein was complexed with 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine yielding reconstituted HDL (rHDL). BAOEC were treated with lipid-free apoAI or rHDL on the apical side (vascular side) for 18 h. The conditioned medium from the basolateral side (sub-endothelial intima side) was incubated with cobalt-Dynabeads to capture His-tag/apoAI. Western blot analysis revealed appearance of apoAI whether added as lipid-free protein or as rHDL. Preliminary tandem MALDI-TOF mass spectral analysis revealed that lipid-free apoAI was more oxidatively modified compared to rHDL. Further studies will focus on identifying the precise sites and nature of modification, other changes to the lipoproteome during transcytosis, as well as alterations in the functional status of the HDL. The long-term goal is to identify direct relationship between structure/composition and changes to the atheroprotective property of HDL as a consequence of transendothelial transport.
This project is supported by NIH grant #GM 105561, McAbee-Overstreet Scholarship and the CSULB/ORSP Student Summer Research Award.
Poster #: 19
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: yeast, growth factors, protein production
Project Title: Recombinant Expression of Epidermal Growth Factor and Fibroblast Growth Factor in Pichia pastoris for commercial production
Author List:
James, Dawn; Graduate, Chemistry, California State University, Fresno, Presenting Author
Tanner, Christopher; Undergraduate, Chemistry, California State University, Fresno
Brooks, Cory; Faculty, Chemistry, California State University, Fresno
Abstract: Epidermal growth factor (EGF) and Fibroblast growth factor (FGF) are commercially important proteins that have made their mark on the beauty industry as a form of modern medicine. EGF levels begin to decrease at the age of 20 after which, over time, the skin gets thinner and the rate at which wounds heal slows down. EGF can induce myriad effects on cells and epithelial tissues, prompting cellular proliferation. FGF is an important anti-aging tool. Currently, commercial production of EGF and FGF occurs through expression of insoluble protein as inclusion bodies in E. coli. The proteins must be then solubilized and refolded in order to be of use. The refolding of protein from inclusion bodies is a long, expensive and tedious process, highlighting the need for alternative means of production. This project aims to produce EGF and FGF in high quantities using the yeast Pichia pastoris as an expression system to ultimately minimize production steps costs for the proteins.
Genes for FGF and EGF were codon optimized for yeast expression and cloned into the yeast expression vector pD912AKS, containing a methanol inducible AOX1 promoter and a metal affinity tag. To facilitate secretion of the proteins to the extracellular medium, the proteins were fused to a alpha-mating factor section signal. We transformed the pD912AKS-EGF and pD912AKS-FGF into P. pastoris by electroporation. Multiple clones were selected and screened for protein expression by western blotting using an His Tag antibody and Anti-FGF antibody. A total of 28 FGF clones were screened, with 6 displaying secreted protein. A total of 10 EGF clones were screened, with 3 showing measurable protein expression. Despite the isolation of several protein-expressing clones, expression yields were too low to be viable for commercial production.
Poster #: 20
Campus: Cal Poly Pomona
Poster Category: Biochemistry
Keywords: purification, microbiome, column chromatography
Project Title: There’s More to Your Gut than Instincts- Investigating the Bacterial Protein BaiH
Author List:
Randle, Laurin; Undergraduate, Chemistry & Biochemistry, California State Polytechnic University, Pomona, Presenting Author
Cao, Amanda; Vanderbilt University, Chemistry
McCulloch, Kathryn; Faculty, Chemistry & Biochemistry, California State Polytechnic University, Pomona
Abstract: Humans are complex organisms that require elaborate biochemical mechanisms to maintain health. One of these mechanisms recycles primary bile acids from the liver to the intestines and then back to liver through enterohepatic circulation. This process allows for nutrients to be absorbed and helps regulate metabolism and homeostasis. However, in the intestines, some natural bacteria interact with the primary bile acids to produce harmful products. One such bacterial pathway catalyzes a 7α-dehydroxylation to produce secondary bile acids, which have been correlated with negative health outcomes such as cancer. This study focuses on a gene, baiH, from a bile acid inducible operon; each gene product of the cluster performs a unique function within the 7α-dehydroxylation pathway. BaiH has previously been demonstrated to perform oxidative reduction chemistry on stereo-specific NAD(H)-dependent 7β-hydroxy-3-oxo-Δ4-cholenoic acids. However, much remains to be learned about the structural basis of enzyme selectivity and specificity. This work utilizes recombinant overexpression and purification of BaiH using E. coli. The baiH gene was first excised and placed in an expression vector, which was inserted into E. coli, a more practical bacterium to work with in a laboratory setting than the producing organism Clostridium hylemonae. Optimized expression conditions were determined to be overnight induction at 16 C once mid log phase was reached. BaiH was then purified using various ion exchange columns and size exclusion chromatography. A HiTrap IEX selection kit with ion exchange columns using a strong cation exchanger, strong anion exchanger, and weak anion exchanger was applied. Each purification using a different resin was followed by size exclusion to analyze oligomeric assembly and SDS-PAGE to analyze purity. The weak anion exchanger provided the best isolation of the protein, possibly due to the exchanger’s ability to interact with the buffer and change pH. The gel analysis supported this strategy, as the purification with the weak anion exchanger was the purest. These findings will be used to scale up BaiH protein purification and may be applicable to similar proteins. This work has been supported by a New Investigator CSUPERB grant and start-up funds to Dr. Kathryn McCulloch.
Poster #: 21
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: apolipoprotein, lipid binding, self-association
Project Title: Probing the lipid binding and self-association properties of apolipoprotein A-I using chimera proteins
Author List:
Patel, Nairuti; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author, Eden Award Nominee
Patel, Nairuti; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract: Human apolipoprotein A-I (apoA-I) is a major component in high-density lipoprotein and plays a critical role in reverse cholesterol transport. The protein (243 residues, 28 kDa) consists of an N-terminal (NT) helix bundle domain and a less structured C-terminal (CT) domain. The CT domain has high affinity for phospholipids and initiates lipidation and formation of discoidal HDL. It is also the site of self-association as the lipid-free protein forms oligomers. However, there is still debate about the exact role of NT and CT helices in initiation of lipid binding and self-association, and the present study aims to gain insight into this. Previously, a chimera composed of insect apolipophorin (apoLp-III) and the CT domain of apoA-I (residues 179-243) acquired high affinity for lipids and self-association properties similar to apoA-I, indicating the feasibility to use chimeras to determine the functional properties of specific regions in apoA-I. In the current study, 3 new apoLp-III/apoA-I chimeras were designed. To study the role of NT helices, residues 1-43 or 44-65 of apoA-I were attached to the NT of apoLp-III. To further define the specific sites in the CT domain, residues 231-243 of apoA-I were attached to the CT of apoLp-III. To prevent lipid binding of apoLp-III, an inactive form of the protein was used. Circular dichroism analysis showed that the chimeras displayed α-helical contents similar to apoLp-III. Phospholipid solubilization rates were high for apoA-I (1-43)/apoLp-III (4.6 x 10-3 s-1) and apoA-I (44-65)/apoLp-III (13.9 x 10-3 s-1), whereas a rate of 0.3 x 10-3 s-1 was obtained for apoLp-III/apoA-I (231-243), similar to apoLp-III (0.4 x 10-3 s-1). Crosslinking with dimethylsuberimidate showed that the three chimeras did not form oligomers. In summary, the structural integrity of apoLp-III was not affected by the addition of apoA-I segments, but none of these were sufficient to cause protein oligomerization. ApoA-I residues 1-43 and 44-65 displayed high lipid binding, indicating a potential role in initiating lipidation of apoA-I. Attachment of residues 231-243 did not enhance lipid binding of the chimera, perhaps because of an insufficient number of amino acid residues to form an amphipathic α-helix. To address this, a 4th chimera was constructed in which the last helix of apoA-I (residues 220-243) was attached to apoLp-III. Functional analysis will reveal if this segment is able to direct apoA-I self-association and binding to lipids.
Poster #: 22
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Apoptosis, Bcl-2 family proteins, Bax
Project Title: Investigating the role of N-terminal domain exposure of Bax in the Regulation of Intrinsic Apoptosis at the Mitochondria
Author List:
Friedlein, Jordan; Graduate, Chemistry, California State University, Fresno, Presenting Author
Dejean, Laurent; Faculty, Chemistry, California State University, Fresno
Abstract: A defining characteristic of cancer cells is the loss of the ability to properly undergo apoptosis, otherwise known as programmed cell death. It has been well known for over two decades that the Bcl-2 protein family, which is made up of both pro and anti-apoptotic proteins plays a major role in regulating the intrinsic pathway of apoptosis. A key member of this protein family, Bax acts as a pro apoptotic effector by translocating from the cytosol to the mitochondria, oligomerizing and forming pores in the mitochondrial outer membrane (MOM). Cytochrome C passes through these pores into the cytosol, irreversibly committing the cell to apoptosis. Because Bax plays such a pivotal role in cell death signaling, it is highly regulated. Bax mediated MOM permeabilization requires the involvement of BH-3 only proteins and protein kinases involved in cell survival regulation. Activation of Bax also requires a substantial change in conformation and of localization of the Bax protein. We know that when Bax is active the N-terminus is exposed. However, the molecular processes linking N-terminus exposure with respect to Bax’s ability to translocate to, insert, and permeabilize the mitochondrial membrane remain to be defined. The P168A mutation is part of a series of mutations at the C-terminus known to activate the Bax protein. Previous research has shown when Bax mutant P168A is expressed in yeast, it has a lower mitochondrial localization than WT Bax. Paradoxically, this mutant has a greater ability to cause cytochrome C release and induce apoptosis. By using an ELISA to probe the recombinant Bax P168A mutant for N-terminus exposure, we have found significantly lower levels of N-terminus exposure in this mutant vs. WT Bax. Taken together, these results suggest that the N-terminus plays a more predominant role in Bax mitochondrial translocation vs. either pore formation or stabilization. Future work will aim to determine the extent of N-terminus exposure of recombinant S184 Bax mutants (i.e. S184A and S184D) to better understand the regulation of Bax pro-apoptotic activity by cell survival protein kinases such as Akt.
Poster #: 23
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: copper, LPP rat, mitochondria
Project Title: Mitochondrial copper binding in LPP rat copper overload
Author List:
Berue, Nathan; Graduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author, Eden Award Finalist
Lichtmannegger, Josef; Institute of Toxicology, Helmholtz Center Munich, Neuerberg 85764, Germany
Linder, Maria C; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Abstract: The LPP rat is a model for human Wilson disease, where the copper “pump” ATP7B is defective and excess copper cannot be excreted in the bile. As shown in this model, the liver toxicosis that occurs results from accumulation of excessive amounts of copper in hepatic mitochondria, causing their destruction. The objectives of the current collaborative studies were to determine the form in which the excess copper was accumulating in these organelles and whether it be the small copper carrier (SCC) identified in the urine and plasma of Atp7b-/- knockout mice. For these studies, mitochondria from the livers of homozygous LPP rats were purified by differential centrifugation, sonicated, and sedimented for 60 min at 100,000 xg. The supernatants obtained had copper concentrations ranging from 10,000-20,000 ppb, 7-8 fold higher than for mitochondrial supernatants from heterozygous rats (which do not have liver toxicosis). Ultrafiltration and size exclusion chromatography revealed that this copper was associated with one major component, and it was much larger than SCC. Incubation with reducing and chelating agents (1mM glutathione, 0.2mM imidazole, 0.2mM iminodiacetate, 0.2mM EDTA) in phosphate buffer pH 7, followed by 3 kDa ultrafiltration, failed to release copper from this component. Methanobactin (0.2 mM), which reduces liver copper content in LPP rats in vivo, also did not directly remove copper from the mitochondrial component. Fractionation on Sephacryl S-300 indicated an apparent molecular weight of less than 50,000. Fractions corresponding to the copper peak were applied to SDS-PAGE. In the case of one fraction with much lower absorbance at 280 nm, only two distinct protein bands (30 and 35 kDa) were observed. Data obtained from mass spectroscopy identified one of these as a plausible candidate for excess copper binding protein in mitochondria, thiosulfate sulfurtransferase (TST). TST, or rhodanese, is a mitochondrial protein largely involved in sulfur metabolism, cyanide detoxification, as well as removal of reactive oxygen species. Western blots of TST showed homozygous LPP rats expressed much higher levels of TST, relative to the heterozygous LPP rats. Immunoprecipitation studies and others are being conducted on both heterozygous and homozygous LPP rats, to better characterize the role of TST in copper toxicosis.
Poster #: 24
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: methanopterin, dihydromethanopterin reductase, methylotroph
Project Title: Optimizing Dephospho-tetrahydromethanopterin (dH4MPT) purification for use in Dihydromethanopterin reductase B (DmrB) assay
Author List:
Aguilar, Edwin; Graduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Rasche, Madeline; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Abstract: Methanogens contain an important one-carbon carrier known as tetrahydromethanopterin (H4MPT) needed for biological methane production. The enzyme that catalyzes the last step of H4MPT biosynthesis is dihydromethanopterin reductase X (DmrX). Inhibition of this O2-sensitive protein has been proposed as a strategy for decreasing methane production as a greenhouse gas. Current obstacles to discovering new DmrX inhibitors include the sensitivity of methanogen cells, H4MPT, and DmrX to destruction by O2. However, the aerobic bacterium Methylobacterium extorquens AM1 contains low concentrations of dephospho-H4MPT (dH4MPT), and other beta-proteobacteria contain an air-stable DmrX homolog (DmrB). To test whether these bacteria may assist in overcoming DmrX limitations, growth conditions of M. extorquens AM1 were modified to increase dH4MPT yield. Feeding the cells with 10 mM succinate midway through the growth cycle extended the exponential growth period, increasing the cell yield from 1 to 3 grams per L of minimal medium. Using centrifugal filtration, ion exchange, and hydrophobic interaction chromatography, dH4MPT was successfully purified and detected using a methenyl-tetrahydromethanopterin dehydrogenase B (MtdB) assay. Subsequently, oxidation conditions for dH4MPT to dH2MPT were tested using the MtdB assay 2, 4 and 6 min after exposure to air at 4°C for 15 seconds with 20 mM beta-mercaptoethanol. This led to an anticipated decrease in dH4MPT of 52%, 67%, and 82%, respectively. However, measurement of dH2MPT using a different assay showed a rate of only 3.3 µM dH2MPT reduced per min, consistent with an unexpectedly low amount of dH2MPT. These data indicate that altering M. extorquens growth conditions successfully increased the amount of cells by three-fold and produced substantial amounts of dH4MPT. However, the subsequent oxidation procedure may have been too long, leading to over-oxidation and a low yield of dH2MPT. The oxidation step may be improved in future experiments by increasing the beta-mercaptoethanol concentration 5-fold and measuring dH2MPT production within one minute after air-oxidation. Optimizing these conditions will allow dH2MPT to be produced as a reliable source and natural substrate for a future DmrB assay, which may be useful in screening for DmrX inhibitors that decrease the production of methane as a greenhouse gas. (Funding was provided by NSF Award #CHE-1508801 and a 2014 CSUPERB Research Development Grant.)
Poster #: 25
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Biochemistry, DNA Repair, fluorescence microscopy
Project Title: The effects of varying DNA flap length from 20 to 50 deoxynucleotides on the mechanism of single-strand annealing in Saccharomyces cerevisiae
Author List:
Fregoso, Fred; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author, Eden Award Finalist
Odango, Rowen; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Camberos Felix, Juan; Graduate, Chemistry and Biochemistry, California State University, Northridge
Fischhaber, Paula ; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract: Mutagens constantly threaten the integrity of genetic material by causing damage in the form of double-strand breaks (DSBs). Such damage is typically repaired through various modes of homologous recombination (HR). This project focuses on a form of HR known as single-strand annealing (SSA), which is triggered when a DSB is located between two DNA repeats. This pathway generates 3’ overhanging single-stranded DNA “flaps” which are cleaved by the Rad1-Rad10 endonuclease, aided by the mediator protein Saw1 in S. cerevisiae. Recent literature has demonstrated that Saw1-dependent recruitment of Rad1-Rad10 to damage sites, and SSA repair proficiency, are both a function of flap length. In this study, we set out to elucidate the precise flap length threshold requiring SAW1 for optimal SSA repair in vivo through fluorescence microscopy and real-time quantitative PCR (qPCR).
In order to investigate the importance of DNA flap length on Rad1-Rad10 and Saw1 recruitment, fluorescently labeled yeast strains were constructed that generate 20, 30, or 50 deoxynucleotide (nt) flaps upon DSB induction. All strains contain 224 tandem copies of the TetO cassette, which bind the constitutively-expressed TetR-RFP protein to allow for the visualization of the DSB site. Yeast strains expressing SAW1 (WT) or deleted of SAW1 (saw1Δ) were analyzed by fluorescence microscopy to observe the recruitment patterns of Rad10-YFP in the absence of Saw1. In addition, WT strains labeled with SAW1-CFP were analyzed to determine if Saw1-CFP and Rad10-YFP simultaneously localize to DSB sites. Results show a decrease in Rad10-YFP recruitment in the absence of Saw1 as the flap lengths increased. Surprisingly, Saw1-CFP recruitment was observed across all flap lengths. Analyses via qPCR were also carried out to quantify SSA repair products formed in WT and saw1Δ strains. The data show diminished repair product formation in the absence of Saw1 as flap lengths increased. Together, these results suggest that Saw1 is required for Rad1-Rad10 recruitment and efficient SSA repair in longer flap substrates with ~20-30 nt representing the key size triggering Saw1-dependence for repair. These findings provide us with a better understanding of one mode of double-strand break repair which, in turn, informs our understanding of cancer pathogenesis.
The authors thank our Department and the NIH SC3 program for funding.
Poster #: 26
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: heat-shock proteins, lipids, mutations
Project Title: Characterization of the relationship between the chaperone and lipid-binding functions of the 70-kDa heat-shock protein, HspA1A
Author List:
Smulders, Larissa; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Saccomanno, Vanessa; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Nikolaidis, Nikolas; Faculty, Biological Science, California State University, Fullerton
Abstract: HspA1A is a stress-inducible seventy-kilodalton heat shock protein (Hsp70) that plays indispensable roles in cellular survival. This protein also localizes at the plasma membrane (PM) of 90% of human tumors and these HspA1A-PM positive cells are radiation insensitive. Although a putative therapeutic would be to inhibit the PM-localization of HspA1A, which is known to be lipid-driven, complete inhibition of HspA1A has detrimental side effects. Therefore, inhibiting the PM-translocation alone seems to be a promising alternative idea. However, the relationship between the chaperone and lipid-binding functions of HspA1A remains unknown. Here, we aimed to elucidate this relationship using a Lysine to Alanine (K71A) mutation, which results in complete loss of the chaperone function. We first determined whether this mutation alters HspA1A’s stability, intracellular and membrane localization, as well as its ATPase and refolding functions using recombinant proteins and ex vivo experiments. Our results revealed that the K71A abolishes both the ATPase and refolding activities, as expected. We then used the lipid vesicle sedimentation (LVS) and Surface Plasmon Resonance (SPR) methods and quantified the binding of the wild-type and mutated HspA1A to several lipids. These experiments revealed that the K71A mutation did not significantly change the binding of HspA1A to lipids under any of the conditions tested. Although these findings strongly suggest that the chaperone and lipid-binding functions are distinct, we were still missing direct evidence and thus decided to perform a series of experiments to directly determine the effect of lipid-binding to the secondary structure and chaperone functions of HspA1A. Specifically, we used Circular dichroism spectrometry, measured the release of inorganic phosphate, and determined the rate of refolding of chemically denatured beta-galactosidase in the presence or absence of particular lipid. These experiments revealed that lipid-binding did not alter the secondary structure of the protein and affected neither the rate of ATP hydrolysis nor the rate of protein refolding and further support the notion that the chaperone and lipid-binding functions of HspA1A do not overlap. These findings provide the basis for future interventions that will inhibit the lipid-driven translocation of HspA1A to the PM of tumor cells making them sensitive to radiation therapy.
Poster #: 27
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Receptor Biochemistry, Transmembrane Signaling, Functional Selectivity
Project Title: Mechanisms of G Protein-Selectivity in Muscarinic Acetylcholine Receptor Family
Author List:
Jaimes, Luis; Undergraduate, Chemistry and Biochemistry; Biology, California State University, Northridge, Presenting Author
Abrol, Ravinder; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract: Muscarinic acetylcholine receptors are integral membrane proteins that regulate many physiological processes. Acetylcholine is a neurotransmitter that binds to the extracellular side of these G protein coupled receptors (GPCRs) activating the Gα subunit of the trimeric G protein inside the cell. There are 5 different muscarinic receptor subtypes (M1-M5), where the same ligand acetylcholine causes M2 and M4 receptors to preferentially couple to the Gαi subunit and the receptors M1, M3, and M5 to preferentially couple to the Gαq subunit. This selectivity of Gα subunits mediates different secondary messenger pathways in the cell: Gαi being responsible for deactivating adenyl cyclase activity and Gαq being responsible for activating phospholipase C. We are using computational biophysical methods to probe the mechanisms of G protein selectivity of this muscarinic receptor family. We have performed 1 microsecond long molecular dynamics (MD) simulations of M1 and M2 receptors coupled to both Gαi and Gαq to obtain relaxed structures of receptor:G-protein complexes and have carried out the thermodynamic analysis of the receptor: G-protein interactions. These studies show that thermodynamically, the wild-type M1 receptor binds stronger to the Gαi subunit and the wild-type M2 receptor binds stronger to the Gαq subunit. This is opposite of the receptors’ G protein preferences observed in the cells. The same analysis for the M2 mutant (M2AALS) with the altered G protein selectivity, which favorably couples to the Gαq subunit in the cells, showed that thermodynamically the M2 mutant also prefers to bind to the Gαq subunit. These data suggest that for the wild-type receptors, the kinetics of receptor:G-protein association might be playing a dominant role over thermodynamics in determining the G protein selectivity. This hypothesis is currently being tested. These studies are beginning to provide us with the missing mechanistic insight into the G protein selectivity of the muscarinic acetylcholine receptors.
Poster #: 28
Campus: Cal Poly San Luis Obispo
Poster Category: Biochemistry
Keywords: Cell-Free Protein Synthesis, Metabolic Engineering, Natural Products
Project Title: Facilitating Intermodular Interactions of a Non-Ribosomal Peptide Synthase Using Communication-Mediating Domains
Author List:
Levine, Max ; Graduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Chacon, Anthony; Undergraduate, Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, Presenting Author
Oza, Javin; Faculty, Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo
Watts, Katharine; Faculty, Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo
Abstract: Epoxomicin is a natural product molecule produced by an Actinobacteria, and its peptide core is biosynthesized by a non-ribosomal peptide synthase (NRPS). A chemically synthesized derivative of this natural product, Carfilzomib, is now marketed as an anti-myeloma drug. The overarching goal of this project is to engineer a biosynthetic system for the synthesis of the Carfilzomib pharmacophore in a bacterial host, in order to replace the expensive chemical synthesis of the drug with a fermentation approach. The only structural differences between the drug core of Carfilzomib and epoxomicin are the identities of 3 amino acids. The NRPS megaenzyme (>500 KDa) that synthesizes the peptide core of epoxomicin has 4 covalently linked modules that each incorporate one amino acid into the molecule. By modifying key amino acid residues in 3 of the 4 NRPS modules, we aim to swap the specificity of these modules, allowing for production of the Carfilzomib core by an NRPS.
Due to the massive size of the NRPS, we have devised a strategy to deconstruct the megaenzyme into 4 separate modules, and then re-capitulate the activity of the NRPS in vitro. The re-capitulation of the megaenzyme is achieved through engineered protein-protein interactions between modules using additional communication mediating (COM) domains. COM domains originate from other wild-type NRPS’s, and have been shown to facilitate interactions between pairs of NRPS modules that do not normally interact. All modules with added COM domains have been successfully cloned using Gibson Assembly with wild-type and modified amino acid specificities. Two modules have been successfully expressed in vivo, and in an E. coli-based cell-free system. Notably, these are among the largest enzymes (>150 KDa) to ever be produced in a cell-free system. To determine how modular the COM domains are in facilitating protein-protein interactions, we are developing an assay with a split green fluorescent protein (GFP), in which an N-terminal and C-terminal region of GFP contain a donor/acceptor pair of COM domains. The capacity of COM domains to support protein-protein interactions is quantified using fluorescence intensity and anisotropy. We will leverage this assay to determine if module-module interactions take place amongst the deconstructed NRPS. Functional assays that monitor product formation between pairs of purified modules will confirm that module activity is maintained. Results from these assays will be presented.
Poster #: 29
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Cancer, Glyoxalase, Glutathione
Project Title: Inhibition of Glyoxalase 1 results in marked increase in intracellular glutathione and reduced cell viability in breast and prostate cancer cell lines
Author List:
Cordova, Benjamin; Graduate, Chemistry & Biochemistry, California State University, Northridge, Presenting Author, Eden Award Nominee
Ahmad, Saadman; Graduate, Chemistry & Biochemistry, California State University, Northridge
Gardon, Nicole; Undergraduate, Chemistry & Biochemistry, California State University, Northridge, Presenting Author
Chan, Linda; Undergraduate, Chemistry & Biochemistry, California State University, Northridge
Matavousian, Arvin; Undergraduate, Chemistry & Biochemistry, California State University, Northridge
Mason, James; Undergraduate, Chemistry & Biochemistry, California State University, Northridge
Bueno, Lisa; Undergraduate, Chemistry & Biochemistry, California State University, Northridge
Kenney, Sydney; Undergraduate, Chemistry & Biochemistry, California State University, Northridge
Anderson, Jennifer; Undergraduate, Chemistry & Biochemistry, California State University, Northridge
Soetyono, Levina; Undergraduate, Chemistry & Biochemistry, California State University, Northridge
Abstract: Glyoxalase 1 (Glo 1) predicts for poor prognosis in prostate cancer patients. It correlates with the TMPRSS2:ERG gene fusion that is a marker for castrate-resistant prostate cancer (CRPC). Glo 1 is also over-expressed in breast tumor tissue relative to adjacent tissue. Glyoxalase 1 and 2 (Glo 1/2) is responsible for the detoxification of reactive carbonyl species that may arise from metabolic processes. The substrates for Glo 1/2 include methylglyoxal and glyoxal. Methylglyoxal is a well-established by-product of prolonged glycolytic flux and glyoxal is generated by lipid peroxidation. Both are reactive carbonyl species and can form adducts with cellular nucleophiles such as DNA, RNA, protein and lipids, leading to diminished cellular function and cell death. The glutathione-dependent Glo 1/2 system converts methylglyoxal to D-lactate. The main objective of our study is to determine the effect of Glyoxalase 1 (Glo 1) inhibition by S-p-bromobenzylglutathione cyclopentyl diester (p-BrBzGSH(Cp)2) and to investigate it as a putative target in castrate-resistant prostate cancer (CRPC) and triple negative breast cancer (TNBC), two difficult to treat cancer sub-types. We have determined the IC50 values in a panel of prostate cancer and breast cancer cell lines using the MTT assay. The IC50 values for the breast cancer cells for the Glo 1 inhibitor, p-BrBzGSH(Cp)2 are as follows: 14.6 μM for MCF-7 cells, 19.5 μM for MBA-MB-231 cells, and 11.7 μM for MDA-MB-468 cells. For the prostate cancer cells, the IC50 values were 11.3 μM for LNCaP, 9.0 μM for DU 145, 14.5 μM for 22Rv1. We quantified glutathione in p-BrBzGSH(Cp)2 treated and untreated cells. We found that p-BrBzGSH(Cp)2 treatment at the IC50 elevates intracellular GSH by 10-50% for all cell lines except for the DU 145 prostate cell line. We have isolated genomic DNA of each cell line to quantify the levels of the DNA adduct, (R)-/(S)-N2-(1-carboxyethyl)-2’-deoxyguanosine ((R)-/(S)-CEdG) in both p-BrBzGSH(Cp)2 treated and untreated cells. We hypothesize that p-BrBzGSH(Cp)2 treatment will result in an elevated level of (R)-/(S)-CEdG adducts versus untreated cells. We are also carrying out the synthesis of the Glo 1 inhibitor, p-BrBzGSH(Cp)2 adapting a previously published method. Taken together, metabolically targeting via Glo 1 inhibition may be part of a viable therapeutic strategy in TNBC and CRPC. [This work is supported in part by a CSUPERB New Investigator Award to Daniel Tamae].
Poster #: 30
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Alzheimer’s disease, butyrylcholinesterase, enzyme inhibitors
Project Title: Synthesis and Characterization of Esters of Fmoc-Amino Acids as Potential Butyrylcholinesterase Inhibitors
Author List:
Pingul, Bianca; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Ramirez, Jennifer; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach
Schwans, Jason; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract: Neurodegenerative diseases such as Alzheimer’s disease (AD) is the sixth leading cause of death in the United States and affects 5.7 million Americans. While cures for this disease have not yet been discovered, several pharmaceuticals are available to alleviate symptoms. These compounds typically target the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Specifically, for individuals with AD it has been found that while AChE activity is slightly decreased or unaffected, BChE activity is increased. The increased BChE activity leads to a depletion of the neurotransmitter acetylcholine, and this depletion is implicated in the progression of dementia. Thus, inhibitors of BChE are sought in the treatment of AD. We previously found Fmoc-amino acids selectively inhibit BChE, leading to a potential new class of cholinesterase inhibitors. Although inhibitory, the Fmoc-amino acids are anionic and the enzyme binds a cationic substrate. We postulated Fmoc-amino acid esters may be better BChE inhibitors, as the ester ablates the negative charge and allows incorporation of substituents that may interact favorably with the enzyme. To test this model, a series of Fmoc-amino acids were esterified using an alcohol and EEDQ (N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline), purified by chromatography, and characterized by NMR and HPLC. The solubility and inhibition properties were evaluated using UV-Vis spectroscopy. To evaluate hydrophobic and cationic-containing compounds, we synthesized leucine and lysine analogs. Although solubility limitations precluded determining inhibition constants (KI values) for leucine, the esters were more potent inhibitors. Whereas a concentration of 100 μM Fmoc-Leu-O- reduced BChE activity ~30%, the same concentration of the methyl ester (Fmoc-Leu-OMe) and ethyl ester (Fmoc-Leu-OEt) reduced activity by ~50%. The inclusion of an ethyl ester on lysine (Fmoc-Lys-OEt) led to an ~40-fold better inhibitor, as Fmoc-Lys-OEt had a KI value of 3.88 ± 0.07 μM compared to 150 ± 10 μM for Fmoc-Lys-O-. Overall, the results suggest esters of Fmoc-amino acids may provide a route for the generation of more potent BChE inhibitors and may guide the design of new Fmoc-containing compounds that specifically and effectively inhibit BChE.
This project is supported by the National Institute of General Medical Sciences of the National
Institutes of Health under Award Numbers R25GM071638, UL1GM118979, TL4GM118980, RL5GM118978, and T34 GM008074.
Poster #: 31
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: NMR , Spider Silk, Isotopes
Project Title: Isotope Labeling Native Spider Silk Proteins with NMR-Active Nuclei for Advanced NMR Characterization of Molecular Structure and Dynamics
Author List:
Villalba, Isaura ; Undergraduate, Department of Chemistry and Biochemistry , San Diego State University, Presenting Author
Soni, Aashana ; Undergraduate, Department of Chemistry and Biochemistry, San Diego State University
Onofrei, David; Graduate, Department of Chemistry and Biochemistry, San Diego State University
Addison, Bennett; Staff, Department of Chemistry and Biochemistry, San Diego State University
Holland, Gregory; Faculty, Department of Chemistry and Biochemistry, San Diego State University
Abstract: Spider silk is a biological polymer used as a template for innovative new materials’ technology development. While the bulk of synthetic spider silk is produced recombinantly, our research is focused on understanding the intermolecular interactions that give native silks their inherent strength which have thus far been unmatched in synthetic fibers produced from recombinant proteins. Understanding these interactions at the atomic level will allow the further development and improvement of silk-based materials. Our research focuses on using Nuclear Magnetic Resonance (NMR) spectroscopy to study spider silk proteins. NMR is a non-destructive and powerful technique for investigating the structure, dynamics, and assembly of biological fibers. Because NMR is only sensitive to isotopes of nuclei having a non-zero spin quantum number, proteins must be enriched with NMR-active nuclei typically, 13C and 15N. Higher isotopic enrichment enables advanced NMR experiments that would otherwise be time-prohibited. Silk protein are large, highly repetitive, and rich in the amino acids alanine, glycine, arginine, and phenylalanine, thus focused our efforts to maximize isotopic enrichment of these residues. The current method involves feeding spiders solutions of concentrated 13C/ 15N-labeled amino acids while forcibly silking them, resulting in 20% isotopic enrichment of the labeled amino acid. Here we present the results of several new and improved labeling techniques. Our first technique looked at under characterized amino acid residues which are commonly found in the repetitive domains including arginine, glutamine, and tyrosine. We measured significant isotopic labeling of these residues with NMR on hydrolyzed silk samples. Using a mixture of labeled and unlabeled amino acids to reduce the amount of salvage and recycling we improved upon previous residue-specific labeling techniques. Additionally we have developed methods to increase isotopic uptake of selective amino acids by incorporating isotopically enriched food sources. This not only gave improved broad labeling of the silk proteins, but allowed us to label residues that are poorly tolerated by direct feeding. By combining efficient isotope labeling techniques with NMR, we are elucidating the molecular interactions responsible for the assembly of spider silk and the resulting mechanical properties.
Poster #: 32
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Biochemistry, DNA Repair, Fluorescence microscopy
Project Title: The effect of distance between repeating DNA sequences on DNA repair pathway and nuclease selection following a double-strand break in S. cerevisiae
Author List:
Camberos Felix, Juan; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Guzmán, Jr., Jimmy; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Mardirosian, Melina; Undergraduate, Chemistry and Biochemistry, California State University, Northridge
Nalbandyan, Linette ; Graduate, Chemistry and Biochemistry, California State University, Northridge
Fregoso, Fred ; Graduate, Chemistry and Biochemistry, California State University, Northridge
Odango, Rowen ; Graduate, Chemistry and Biochemistry, California State University, Northridge
Sanchez, Romny ; Graduate, Chemistry and Biochemistry, California State University, Northridge
Tran, Samuel ; Graduate, Chemistry and Biochemistry, California State University, Northridge
Fischhaber, Paula; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract: DNA double-strand breaks (DSBs) have cytotoxic effects and can lead to cancer. DSB repair pathways include the non-conservative single-strand annealing (SSA) and the conservative synthesis-dependent strand annealing (SDSA) pathways. DSBs flanked by DNA repeats are preferentially repaired by SSA in S. cerevisiae, but the effect of repeat distance and degree of repeat sequence homology on the preference for SSA are not well defined. Saw1 recruits the endonuclease Rad1-Rad10 to cleave 3′ overhanging single-stranded DNA flaps formed during SSA. Existing evidence suggests that short flaps are instead resected by polymerase delta (Pol δ). This work investigated the effects of repeat distance and homology on the preference for SSA and the method of flap removal.
To test the importance of DNA repeat distance and homology on the preference for SSA, yeast strains were cloned containing two imperfect DNA repeats flanking an inducible DSB site that were separated by stretches of 10,000 deoxynucleotides (nt). The DSB site was labeled by binding TetR-mRFP to 224 copies of the TetO DNA site and Rad10 was labeled with yellow fluorescent protein (Rad10-YFP). Recruitment of Rad1-Rad10 to the DSB site after DSB induction was monitored by counting colocalized RFP/YFP foci observed in fluorescent images. To discern SSA from SDSA repair events, strains were either wild-type in, or deleted of RAD51 and RAD52. Rad52 participates in both SSA and SDSA but Rad51 is only required for SDSA. Results showed that only Rad52, was required to observe YFP/RFP colocalized foci. We conclude that yeast prefers the SSA pathway over SDSA even when DNA repeats are as distally-situated as 10,000 nt and only 80% homologous.
To observe the effect of repeat distance on whether Pol δ or Rad1-Rad10 removes 3′ flaps during SSA, we cloned strains in which repeats were ~100 nt apart and mutant in POL δ or both POL δ and SAW1. Repair product formation at time points up to 2 hours after DSB induction was quantified using quantitative Polymerase Chain Reaction. Preliminary data show that SSA repair products are formed similarly in POL δ mutants and wild-type strains, while the POL δ SAW1 double mutant shows a marked decrease in repair products. The data suggest that 50 nt flaps do not require Pol δ, but are fully dependent on Saw1, for cleavage. Overall, these results increase our understanding of SSA pathway selection which could aid in finding better strategies for cancer prevention and therapy.
Poster #: 33
Campus: San José State University
Poster Category: Biochemistry
Keywords: cancer, intracellular pH, signaling
Project Title: Identifying pH Sensitive Tumorigenic Proteins Using A Genetic Screen in Drosophila melanogaster
Author List:
Orozco, Daniel; Graduate, Biological Sciences, San José State University, Presenting Author
Grillo-Hill, Bree; Faculty, Biological Sciences, San José State University
Abstract: Normal cells maintain their intracellular pH (pHi) at a constant physiological level near neutral. Recently, regulated changes in pHi have been observed during normal cellular processes, including programmed cell death, differentiation, and proliferation. Dysregulated pHi is present in diseases including cancer, neurodegenerative diseases and lysosomal storage diseases and is thought to contribute to disease pathology. To determine role for dysregulated pHi in diseases, we generated transgenic flies that inducibly express the Na-H exchanger Dnhe2 in the developing Drosophila eye. Over-expressing Dnhe2 in the eye causes a rough eye phenotype eye, which we used as a background for a dominant modifier screen to identify pH-regulated proteins. We obtained 215 lines of flies, each with a different deletion in the second chromosome, and crossed them to flies overexpressing DNhe2. Genetic interactions were classified as enhancers (eyes look more rough) or suppressors (eyes look more like control). We used genetic analysis to identify the pathways and individual genes responsible for genetic interactions. One exciting candidate pH sensitive gene identified in this screen is jellybelly (ieb). Loss of jeb enhanced the DNhe2 phenotype, such that the eye appears to lack any structure, which we term a glazed phenotype. Jeb is a ligand that activates the oncogenic protein Anaplastic Lymphoma Kinase (ALK) in a variety of tumors, including non-small-cell lung cancer and anaplastic large cell lymphomas. In normal cells, ALK is required for differentiation of several distinct lineages including muscle, specific retinal cells and enteric neurons in gut muscles. Further study of Jeb will define its roles in normal and cancer cells, and help us understand regulation by pH mediates its functions during development and disease.
Poster #: 34
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: Splicing, RNA binding proteins, Phosphorylation
Project Title: Determining the Role of Phosphorylation in RNA Binding Protein Function
Author List:
Pina, Jeffrey; Graduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author, Eden Award Finalist
Keppetipola, Niroshika; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Abstract: The process of alternative splicing is regulated in part by trans-acting RNA Binding Proteins that bind to corresponding cis-elements on the pre-mRNA and modulate spliceosome assembly to either include or exclude a regulated exon from the gene transcript. More than 90% of human multi-exon pre-mRNA transcripts undergo alternative splicing and dysregulation of this process is associated with many neurodegenerative diseases underscoring the importance of alternative splicing regulation. One such family of RNA binding proteins consists of polypyrimidine tract binding proteins PTBP1 and PTPB2. PTBP1 is expressed nearly ubiquitously except in neurons and myocytes. In neurons, PTBP1 is replaced by PTBP2. The amino acid sequence of PTBP2 is 74% identical to that of PTBP1. The two proteins share a similar domain organization, and recognize and bind to the same sequence elements adjacent target exons. However, PTBP2 is a significantly weaker repressor than PTBP1 of several neuron specific exons. Neuronal progenitor cells express PTBP1; but during differentiation, the level of PTBP1 is down-regulated, and that of PTBP2 is up-regulated. These changes in PTBP protein expression alter the splicing of a set of neuronal exons that are more sensitive to PTBP1, leading to changes in many transcripts that code for proteins critical for development and maturation. How these paralogs elicit these distinct splicing outcomes is completely unknown. Recently, we conducted mass spectrometry analysis of PTBP1 and PTBP2 and discovered the two proteins have distinct chemical modifications. Our results highlight that the two proteins are phosphorylated and that PTBP2 has many non-overlapping distinct sites of phosphorylation than PTBP1. Thus, we hypothesize that reversible phosphorylation of PTBP2 dictates its neuron-specific splicing activity. To test this, we conducted electrophoretic mobility shift assays with PTBP2 in the presence and absence of ATP to assay a direct role of PTBP2 phosphorylation in RNA binding affinity. Preliminary data highlight that phosphorylation of PTBP2 leads to lower RNA binding affinity. We also conducted mass spectrometry analysis of PTBP2 to identify phosphorylation-dependent partner proteins that may play a role in its splicing activity. We are currently analyzing the data from this assay. Collectively, our studies will answer fundamentally important questions about how structurally related paralogous proteins dictate different splicing outcomes
Poster #: 35
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: zinc metalloprotease, small molecule inhibitor, botulism
Project Title: Designing peptidomimetic inhibitors for the Botulinum Neurotoxin Type A Light Chain
Author List:
Thompson, Jordan; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton, Presenting Author
Palomino, Alejandra; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton, Presenting Author
Rodriquez-Beltran, Sandra; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton
Salzameda, Nicholas; Faculty, Chemistry & Biochemistry, California State University, Fullerton
Abstract: The botulinum neurotoxin (BoNT) is the most toxic protein known to man causing the neuroparalytic disease, Botulism. The neurotoxin consists of a heavy (HC) and light chain (LC) that work together to cause paralysis in humans. Once humans are infected with the toxin, the HC attaches to nerve cells and injects the LC into the cell. The LC is a zinc metalloprotease that cleaves SNARE proteins required for neurotransmission. Once a SNARE protein is cleaved, neurotransmission is halted resulting in flaccid paralysis. Cleavage of a SNARE protein is irreversible and in severe cases result in death or long recovery times. There is an urgent need for therapeutics to counter the deadly effects of BoNT intoxication. The potency and ease of production perpetuate BoNT as a possible bioterrorism weapon. In the event of a biological attack, current treatments are not adequate for widespread infections. New treatments against BoNT are urgently needed. A therapeutic avenue for treatment of botulism is inhibition of the BoNT LC by small molecule inhibitors.
The goal of our research is to design novel therapeutics to inhibit the BoNT LC. This goal was accomplished by synthesizing molecules that mimics peptide bonding to the BoNT LC active site, thereby disrupting enzymatic activity. The inhibitor scaffold contains an isoleucine functionalized with a hydroxamic acid on the C-terminus and a biphenyl connected to the N-terminus by a sulfonamide linker. A library of compounds was synthesized to probe the importance of the biphenyl, sulfonamide and isoleucine for inhibition of the BoNT LC.
The compounds were evaluated as inhibitors by an enzymatic assay. The inhibition study revealed the biphenyl, sulfonamide and amino acid are important for inhibition, as changes to these domains greatly affected inhibition. An electrostatic contact was identified between a chlorine at C-2 of the biphenyl and the active site. The sulfonamide linker was central to inhibition as modifying this linker to an amide or amine greatly reduced inhibition. BoNT LC inhibition was also sensitive to alterations in the stereochemistry of the amino acid. The D-isoleucine stereoisomer with the sulfonamide linker increased inhibition. A 2nd generation compound was synthesized based on these results and has a measured IC50 value of 587 nM for the BoNT LC. Overall, the inhibition data reveals that the scaffold has unique binding interactions and is a promising therapeutic lead against botulism.
Poster #: 36
Campus: CSU Los Angeles
Poster Category: Biochemistry
Keywords: breast cancer, protein structure, cysteine
Project Title: Analysis of cysteine thiol surface accessibility in t-Darpp chemoresistance protein
Author List:
Aldana-Mendoza, Jesus; Graduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Farias, Phillip; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles
Magdziarz, Patrycja; Staff, Chemistry and Biochemistry, California State University, Los Angeles
Katz, Benjamin; University of California Irvine
Momand, Jamil; Faculty, Chemistry and Biochemistry, California State University, Los Angeles
Abstract: Approximately 12% of women in the US will contract breast cancer during their lifetime. If detected at a late stage, breast cancer response to chemotherapy can be variable. For Her2-type cancers, treatment with the antibody drug Herceptin adds approximately one year of cancer-free survival to the patient but, almost invariably, the cancers acquire resistance. One mechanism of resistance is by overexpression of t-Darpp, a protein that activates protein kinase A and AKT. Drugs targeting t-Darpp may prove to benefit breast cancer patients but efforts to develop such drugs are hampered by the lack of protein structural information. To gain insight into the structure of t-Darpp we sought to determine if its Cys residues are located on the surface of the protein or buried. Cysteines are frequently required to maintain protein structure. In t-Darpp, its two cysteines, Cys 36 and Cys 119, are evolutionarily conserved and therefore may play important roles in t-Darpp structure and function. We used cysteine modification reagents 5,5′-dithiobis-(2-nitrobenzoic acid) and methoxypolyethylene glycol-maleimide (Mal-PEG) on recombinant t-Darpp to explore cysteine thiol surface accessibility. Treatment with 5,5′-dithiobis-(2-nitrobenzoic acid) yielded an average ratio of only 0.12 free thiol groups per t-Darpp polypeptide. Treatment with Mal-PEG yielded no supershifted bands on SDS-PAGE. To rule out the possibility that t-Darpp thiol groups were oxidized, t-Darpp was digested with chymotrypsin and masses of its peptide products were measured by Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry. Two peaks of 1163.6 Da and 1739.9 Da were observed that are consistent with peptides containing reduced Cys 36 and reduced Cys 119 respectively. Peaks with masses corresponding to common oxidized forms of cysteine were below detection level. Our data is consistent with the hypothesis that the thiol residues of Cys 36 and Cys 119 are buried in the protein.
Poster #: 37
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: manganese, oxidation, peroxidase
Project Title: Detection of covalent heme binding in a bacterial manganese-oxidizing protein
Author List:
Li Qian, Keizen; Graduate, Biological Science, Presenting Author
Johnson, Hope; Faculty, Biological Science, California State University, Fullerton
Abstract: Microbially-mediated manganese (Mn) oxidation facilitates global bioavailability of nutrients including iron, sulfur, and carbon and may have applications for bioremediation of organic and heavy metal pollution. Despite the identification of several bacterial manganese-oxidizing enzymes, their mechanisms are incompletely characterized. The catalytic domain of MopA, a heme-dependent manganese oxidase from a marine alphaproteobacterium, has been heterologously expressed and partially purified as MopA-HP. MopA-HP has sequence similarity to heme peroxidases such as lactoperoxidase (LPO) which autocatalyze covalent bond formation with heme. Covalent bonds with heme are implicated in the ability of LPO-like heme peroxidases to perform high-redox-potential reactions such as bromide oxidation, or in the case of MopA-HP, Mn(II) oxidation. Although MopA-HP purifies as an apoenzyme, when incubated with heme, in-gel heme staining identified heme in the molecular weight band containing MopA-HP, consistent with covalent attachment of the heme to MopA-HP. Two amino acids known to bind heme covalently in LPO are conserved in MopA-HP and were targeted for site-directed mutagenesis: D206 and E472 were substituted with alanine. E472A was compared with wild-type (wt) protein using absorbance spectroscopy and in-gel heme staining. Loss of E472 suppressed redshift (Soret peak at 389 nm vs 408 nm in wt) but not in-gel heme staining or Mn oxidation activity. Future experiments with the D206A and D206A/E472A mutants will show whether MopA-HP covalently binds heme at these sites to enable the high-redox-potential reaction.
Poster #: 38
Campus: Stanislaus State University
Poster Category: Biochemistry
Keywords: DNA damage, toxicology, aldehyde
Project Title: Hookah and Vaping: Safe Nicotine Delivery Systems?
Author List:
Bethishou, Britiel; Undergraduate, Biology, California State University, Stanislaus, Presenting Author
Dunham, Rolaun; Undergraduate, Kinesiology, California State University, Stanislaus, Presenting Author
Skochko, Alex; Undergraduate, Chemistry, California State University, Stanislaus
Stone, Koni; Faculty, Chemistry, California State University, Stanislaus
Abstract: Hookah and vaping have recently been popularized as alternative to smoking tobacco. Hookah heats tobacco with a very hot piece of charcoal and then the air is pulled through the hot tobacco, this smoke is then pulled through water before it reaches the user’s mouth and lungs. Vaping uses electricity to vaporize a propylene glycol/glycerine matrix that contains nicotine and flavoring agents. This vapor is then pulled directly into the mouth and lungs. The mainstream vapors from both hookah and vaping were collected on Cambridge filters, the filters were soaked in water and then the aqueous solutions were dried via vacuum centrifugation. After determining dried mass, each sample was reconstituted with water and then analyzed by UV spectroscopy; the hookah samples had absorbance at 275 nm. Our original hypothesis was that the water soluble tar components would be trapped in the water that the hookah smoke is pulled through. Thus, there should have been no absorbance at 275nm. The vaping samples did not absorb at 275; since vaping does not burn or heat tobacco, we did not expect to find the same absorbance as the cigarette tar radical. Gel electrophoresis was used to analyze the nicking of plasmid (pUC18) DNA. Aqueous solutions of Hookah and vaping solutions were tested in our DNA nicking assay. Briefly, we use supercoiled pUC18 DNA, this DNA unwinds after a single nick. The supercoiled and nicked (open circular) DNA migrate as two distinct bands in the agarose gel. The density of each band is then quantitated to determine the amount of DNA nicking. The amount of nicking by both vaping and hookah solutions is orders of magnitude less that the nicking we have detected with cigarette smoke. However, we have detected aldehydes in both vaping and hookah solutions. Aldehydes are known to be toxic, primarily by their facile reaction with primary amines and thus their ability to wreck havoc with protein structure. We have implemented an assay to detect aldehydes in both hookah and vaping solutions. The reagent, Purpald, 4-Amino-3-hydrazino-5-mercapto-1,2,4-triazole, readily reacts with aldehydes and then the oxidized product of the reaction produces purple solutions. Then, we analyzed the purple solutions by UV/VIS. Based on the UV spectra, we have detected acetaldehyde in both vaping and hookah solutions.
Poster #: 39
Campus: CSU East Bay
Poster Category: Biochemistry
Keywords: apolipoprotein A-I, lipoprotein, fluorescent
Project Title: Synthesis of Model High Denisty Lipoproten Particles for Examining the Relationship Between HDL Particle Size and Apolipoprotein A-I Exchange Rate
Author List:
Erhahon, Heinrich; Graduate, Chemistry & Biochemistry, California State University, East Bay, Presenting Author
Oda, Michael; Childrens Hospital Oakland Research Institute
Borja, Mark; Faculty, Chemistry & Biochemistry, California State University, East Bay
Abstract: Objective: High density lipoprotein (HDL) is a particle involved in cholesterol regulation, and high levels of HDL are associated with reduced risk of cardiovascular disease. It exist in several different sizes. The major protein component of HDL is apolipoprotein A-I (apoA-I). Our aim is to determine whether or not there is a functional difference between HDL particles of different sizes by examining the rate of exchange of apoA-1 on several sizes of laboratory-ssynthesized model HDL particles.
Method: Recombinant human ApoA-1 modified with single cysteine mutations were expressed in bacteria (E. coli) and labeled with Alexa 488 and 647 fluorophores. Model HDL particles of defined size were created by varying protein:lipid ratio using phosphatidyl choline and cholesterol as lipids, and apoA-I labeled with Alexa 647 as protein.
Results: The recombinant proteins were purified. Alexa 488 and 647 fluorophores were attached to the proteins. 10-17nm HDL particles were synthesized.
Conclusion: We have successfully synthesized a panel of HDL particles in the 10-17 nm size range. The next steps will be to perform exchange assays to determine the rate of exchange.
Poster #: 40
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: antibody, immunotherapy, cancer
Project Title: Humanization of a MUC16 Specific Monoclonal Antibody for the Treatment of Pancreatic Cancer
Author List:
White, Brandy; Graduate, Chemistry, California State University, Fresno, Presenting Author, Eden Award Finalist
Broosk, Teresa; Faculty, Chemistry, California State University, Fresno
Brooks, Cory; Faculty, Chemistry, California State University, Fresno
Abstract: Pancreatic ductal adenocarcinoma is a highly aggressive and lethal form of cancer with an extremely low survival rate. The near death sentence associated with a diagnosis of pancreatic cancer is associated with the failure of traditional therapeutic approaches to combating the disease. The clinical success of therapeutic antibodies for cancer treatment is now well established, however there are no approved antibody therapies for pancreatic cancer. Here we report the humanization of a therapeutic antibody, which in mouse models halted the progression and metastases of pancreatic cancer. The antibody binds MUC16, a membrane glycoprotein that is overexpressed and abnormally glycosylated in pancreatic cancers. The antibody was humanized by grafting the antigen-binding CDR loops onto a human germline antibody scaffold. Residues from the mouse sequence located in the vernier zone predicted to be important for the structure of the CDR loops were maintained in the humanized antibody. Transient transfection of the humanized antibody in expiCHO cells resulted in a yield of 1 mg per ml of culture supernatant. ELISA binding assays demonstrated that the humanized antibody bound to MUC16 with a comparable affinity to that of the parent mouse antibody. Epitope mapping revealed that the antibody bound to an SEA domain within the tandem repeat region of MUC16. Finally, a high resolution X-ray structure of the humanized antibody Fab region was determined to a resolution of 1.9 Å. The structure reveled a light chain CDR1 with an extended conformation, and a binding pocket for the MUC16 SEA domain. The structure also illuminated the importance of vernier zone residues in maintaining the CDR loop conformations. The x-ray structure will guide the development of a 2nd generation of humanized antibody. The results presented here pave the way for future engineering of this novel antibody for the treatment of pancreatic cancer.
Poster #: 41
Campus: San Francisco State University
Poster Category: Bioengineering
Keywords: electromyography, pattern recognition , feature extraction
Project Title: Evaluating Computationally-Efficient Spatial Features in sEMG Array-Based Gesture Recognition
Author List:
Phan, Justin; Undergraduate, School of Engineering, San Francisco State University, Presenting Author
Zhang, Xiaorong; Faculty, School of Engineering, San Francisco State University, Presenting Author
Abstract: Surface electromyography (sEMG) is a technique for measuring biosignals produced by contracting muscles using surface electrodes. sEMG signals contain neuromuscular information and can be analyzed using pattern recognition (PR) algorithms to identify movement intents. sEMG PR has shown great promise in allowing intuitive muscle control of prosthetics and assistive robots. This project focuses on evaluating the effectiveness of a newly developed computationally-efficient spatial feature set, which analyze the intensity and structure of the signals recorded from equally spaced sEMG arrays and evaluate the similarities between adjacent electrodes, in real-time sEMG PR for gesture recognition. The proposed feature set includes a scaled mean absolute value (SMAV) feature scaling out the overall gesture intensity from individual channels and a set of adjacent features (AF) representing adjacent uniqueness and influence of adjacent electrodes. Offline experiments on sub-datasets in the open-source NinaPro Database, from the second dataset (DB2) to the sixth (DB6), used sEMG data from 52 finger, hand, and grasping motions. Data were sampled at 2000 Hz, except DB5 sampled at 200 Hz, and collected from 10 to 40 able-bodied subjects, except DB3 consisting of transradial amputees. Two PR algorithms were tested, including Linear Discriminant Analysis and Support Vector Machines. The proposed spatial features, and two commonly used feature sets, including the Hudgin’s time-domain (TD) features and 6th order autoregression (AR), were extracted from the sEMG signals. The computational complexity of the spatial feature set is comparable to Hudgin’s TD features and much lower than the AR features. Averaged PR accuracies showed that the spatial feature set outperformed TD and AR feature sets by 4-7% for DB5. For datasets with high sampling frequency, the combination of SMAV and AR features generally performed best with a PR accuracy of 1-12% higher than the second best-performing feature configuration. The results suggest that the SMAV feature can improve sEMG PR accuracy in almost all conditions and the AF set can potentially improve sEMG PR accuracy for data with low sampling frequency while reducing the system’s computational-complexity for real-time operation. Research on a wider variety of datasets and PR algorithms is currently being conducted.
This project was supported by the National Science Foundation (NSF #1752255).
Poster #: 42
Campus: San José State University
Poster Category: Bioengineering
Keywords: prosthesis, gait, simulation
Project Title: A 3D computational model for evaluating muscle recruitment in above-knee prosthesis users
Author List:
Aguilar, Robert; Graduate, Biomedical Engineering, San José State University, Presenting Author
Antar, Mohamed; Undergraduate, Biomedical Engineering, San José State University, Presenting Author
Leineweber, Matthew; Faculty, Biomedical Engineering, San José State University
Abstract: Prosthetic devices are used to restore mobility in patients with missing limbs due to congenital conditions or amputation. However, these devices cannot fully replicate the function of the missing body parts of these patients. As a result, prosthesis users must adapt their movements to compensate for pain, decreased range of motion, or diminished sensory perception. This study aims to design a three-dimensional computational model to simulate walking for both able-bodied individuals and prosthesis users. The inverse dynamics model was constructed using OpenSim, and is driven using able-bodied gait kinematics. The model calculates the muscle forces required to produce the measured kinematics, both with and without a knee prosthesis. Special attention is paid to the muscle forces driving motion about the hip of the prosthesis-side leg. Kinematics data were collected from ten participants using a wearable inertial-measurement-unit (IMU) based motion capture system. The IMU sensors were attached to the head, sternum, shoulders, upper arms, lower arms, hands, pelvis (over the sacrum), upper legs, lower-legs, and on the feet of each participant. Recorded kinematics include joint angles, body-segment orientations, and body position. Ground reaction forces were also calculated from pressure sensors embedded in the participants’ shoes. Each participant walked a total of ten steps in three different speeds (slow, normal, fast). Three runs were performed for each speed, and three consecutive steady-state strides were selected from each trial to be input to the model. Preliminary results have calculated muscle forces for the able-bodied participants, and integration of the prosthesis into the modified able-bodied model has proven feasible. Future work will compare the results of the prosthesis model against the able-bodied baseline to identify differences in muscle forces required to produce typical gait patterns with a prosthetic device. This model will ultimately be a valuable tool for informing more effective clinical rehabilitation techniques and improving future prosthesis design.
Poster #: 43
Campus: Cal Poly San Luis Obispo
Poster Category: Bioengineering
Keywords: Diagnostic devices, Microfluidic paper device, HIV-1
Project Title: A Paper-Based Point-of-Care Diagnostic Device for the Detection of Human Immunodeficiency Virus (HIV-1) Particles
Author List:
Strong, Brandon; Graduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author, Eden Award Nominee
Tod, Nicholas; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Lore, Brittany; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Escamilla, Emiliano; UCSD, California Polytechnic State University, San Luis Obispo
Mercado, Oscar; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Martinez, Andres; Faculty, Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo
Martinez, Nathaniel; Faculty, Biological Sciences, California Polytechnic State University, San Luis Obispo
Abstract:
Objective: To develop a cost-effective microfluidic paper-based analytical device (MicroPAD) capable of HIV biomarker quantitation at the point of care without the use of sophisticated supporting equipment.
There is a widely recognized need for improving the timeliness of initiating antiretroviral therapy (ART) following HIV diagnosis. While laboratory tests are the current gold standard for HIV diagnosis, point-of-care (POC) HIV-1 tests can provide patients with rapid results. However, current POC devices on the market are often expensive, fail to detect acute infections due to low sensitivity, and only offer binary assessments of analytes of interest.
MicroPADs are increasingly utilized as rapid diagnostic tests in field and POC settings. Paper-based substrates are an ideal fabrication medium in diagnostics applications due to inherent characteristics such as, low cost, robustness, ease of use, and the ability to operate without sources of power. While some quantitative microPADs are available, many require supporting equipment.
We have developed a novel 3D microPAD capable of autonomous chronometric quantitation at low picomolar concentrations via enzymatic degradation of a metastable gelatin biomatrix. Four paper layers were fabricated via wax printing and stacked to create 3D flow-through channels bounded by hydrophobic wax barriers. Enterokinase (EK, activator enzyme) was applied to the loading inlet (layer 1), which catalyzed the activation of trypsinogen (zymogen) to trypsin (non-specific serine protease) (layer 2). Trypsin then cleaved the gelatin biomatrix (layer 3) in a concentration-dependent manner. Elapsed time between sample addition to the inlet and presence in the collection well (layer 4) following biomatrix penetration was measured with sensor electrodes connected to a microcontroller. Time was found to be inversely proportional to EK concentration (R2=0.98). We found the device to be sensitive to as low as 7 femtomoles of activator enzyme. The biomatrix was subsequently characterized under a range of potential environmental conditions and displayed high stability at a range of temperatures (20-28C), humidities (20-90%), ph (1-11.5), and over time (0-12 months). We are now working on conjugating EK to an anti-P24 antibody (HIV-1 biomarker used in 4th generation POC tests) to create an immunoassay. This technology could allow for the rapid and cost-effective diagnosis of HIV, as well as rapid temporal monitoring of HIV-1 viral load.
Poster #: 44
Campus: CSU San Marcos
Poster Category: Bioengineering
Keywords: Stem Cells, Mitochondria, Cytoskeleton
Project Title: Expression and Localization of Mitochondria in Human Adipose-Derived Stem Cells is Affected by Substrate Stiffness
Author List:
Burch, Shalise ; Graduate, Biological Sciences, California State University San Marcos, Presenting Author
Javier, Areli J.; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Kumar, Rosaline; Undergraduate, Biological Sciences, California State University San Marcos
Day, Holly; Undergraduate, Biological Sciences, California State University San Marcos
Luna Lopez, Carlos ; Faculty, Biological Sciences, California State University San Marcos
Abstract: Mitochondria are long deemed as the powerhouses of the cell, where ATP is produced. In stem cells, research has shown that mitochondrial dynamics play a critical role in proliferation, self-renewal and cell-signaling, thus indicating that they serve roles beyond cellular respiration.
The movement and distribution of mitochondria depend on the cytoskeleton network. Stem cells modify this network in response to the different mechanical microenvironments. Therefore, we hypothesized that the distribution of mitochondria inside stem cells would be affected by cell-substrate interactions.
We used human adipose-derived stem cells (HADSCs) and created substrates of 1 MPa stiffness using polydimethylsiloxane (PDMS). We used 1 GPa glass substrates for comparison. Substrates were coated with Poly-d-lysine for 30-min to promote cell adhesion. We plated HADSCs and waited 24-hours before imaging. To live-stain mitochondria, we used MitoTracker Green FM with a 45-min incubation. We imaged cells (50 cells, 6 independent experiments) using a fluorescence microscope (LS560 Etaluma) placed inside a 37 degrees C, 5% CO2 incubator. We used phase-contrast micrographs to analyze the cell area, 488 Phalloidin to stain the actin cytoskeleton and 1-hour time-lapses over 4 days to analyze cell division. We analyzed cell area and total cell fluorescence of MitoTracker using ImageJ (NIH). For statistics between two sets of data we used an unpaired t-test with Welch’s correction.
We found that HADSCs on PDMS have ~30% less cell area and fewer actin fibers compared to glass substrates (p<0.03). Interestingly, we also found that the average rate of cell division was 35% lower on PDMS substrates (p<0.02). We found that the mitochondria of cells on PDMS substrates were localized primarily around the nucleus. In contrast, for cells plated on glass we found the mitochondria localized both in the center and edges of the cells, including at the cell protrusions. Mitochondria of cells on glass could be localized as far as the tips of cell protrusions.
These results indicate that the distribution of mitochondria is affected by substrate stiffness, an effect that could be correlated to the observed decreased in cell area and actin stress fibers. In the future, we will investigate what is the role of mitochondria at the protrusions of HADSCs. We will also explore how the change in distribution caused by the mechanical microenvironment affects differentiation and stem cell signaling.
Poster #: 45
Campus: Cal Poly San Luis Obispo
Poster Category: Bioengineering
Keywords: Microfluidics, Cell Culture Optimization, Electrical Impedance Spectroscopy
Project Title: Platform for multiplexed, on-chip microfluidic cell culture optimization with automated image acquisition and impedance monitoring
Author List:
Foley, John; Graduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Myers, Andrew; Undergraduate, Mechanical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Shavey, Gavin; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Hawkins, Benjamin; Faculty, Biomedical Engineering and Electrical Engineering, California Polytechnic State University, San Luis Obispo
Abstract: While cell culture is often routine, optimizing cell culture often requires time-intensive and exacting experimental effort. Modification of the cell genotype, cell co-culture, and other scientifically interesting alterations disturb the balance of media components and culture conditions, leading to poor cell outcomes. We developed a platform for cell culture optimization using a microfluidic chip containing 16 culture wells – each fed with varying concentrations of media components – and an automated process for image acquisition and measurement of cell electrical properties.
The microfluidic device includes a gradient generator that mixes two inlet fluids and feeds 16 culture wells a linearly varying concentration. Microfluidic channels were fabricated in poly(dimethylsiloxane) (PDMS) from a master mold consisting of SU-8 photoresist on a silicon wafer. The PDMS channel was closed using a glass slide patterned 16 interdigitated electrode arrays aligned with each culture well. COMSOL software was used to verify the design computationally. Experimentally, 1mM fluorescein in DI water and pure DI water were introduced to the two inlets to verify the concentration distribution. Average fluorescence intensity in each culture well was captured using an inverted fluorescence microscope and calculated using ImageJ image analysis software. The distributed electrical impedance of each culture well as a function of frequency was measured using a digital oscilloscope and automated using LabView. A custom circuit board was integrated to make connection to all cell culture chambers and signals were routed to the oscilloscope using an Agilent Instrument Driver with a Multiplexing RF Switch. To validate electrical measurements, the impedance of DI water and 0.1% w/v sodium chloride were tested on chip. Temperature was controlled using a custom designed and fabricated microscope stage, cartridge heater, and PID controller.
NIH 3T3 fibroblasts were seeded in the device and growth was monitored by automated fluorescence microscopy and impedance spectroscopy to demonstrate the effects of reducing cell media concentration on cell health. Evaluation of cell metabolic health under varying culture conditions is the key to determining the unique requirements of complicated cell system interactions. Automating the measurement process reduces workload, experimental error, and time required to optimize cell culture conditions.
Poster #: 46
Campus: Cal Poly Pomona
Poster Category: Bioengineering
Keywords: titanium, dental, electrochemical
Project Title: Corrosion Studies of a new class of titanium alloys for dental applications
Author List:
Benoun, Jacob; Graduate, Chemical and Materials Engineering, California State Polytechnic University, Pomona, Presenting Author
Pacheco, Carlos; Undergraduate, Chemical and Materials Engineering, California State Polytechnic University, Pomona
Bae, Jaewan; Undergraduate, Chemical and Materials Engineering, California State Polytechnic University, Pomona
Ravi, Vilupanur; Faculty, Chemical and Materials Engineering, California State Polytechnic University, Pomona
Abstract: Titanium and its alloys have seen widespread use in the field of dentistry, with seven different types used in dental implants. The most popular alloy is Ti-6Al-4V wt.% (Ti64) which strikes a balance between elastic modulus, strength and corrosion resistance; however, some reports have shown vivo exposure to aluminum and vanadium ions can lead to negative health effects. In addition, in this alpha+beta alloy, the alpha phase has poor corrosion behavior versus the beta phase, leading to the possibility of early failure. Previous research conducted by our group has indicated the existence of a voltage gradient between these phases, supporting these findings. In the current research, two types of alloy systems were investigated, i.e., the Ti64 series with boron additions and a novel 𝛽-phase alloy, Ti-28Nb-20Zr wt.% (Ti2820). The latter has sparked interest as a potential dental implant material due to its non-toxic constituents and lower elastic modulus, while the former provides a baseline to study the effect of B additions to Ti alloys. Three coupons (N = 3) of each alloy, cylindrical in form, 6.0 ± 0.2 mm long and with a diameter of 13 ± 0.1 mm, of Ti64, Ti64-0.01B, Ti64-0.04B and Ti2820 were tested in Phosphate Buffered Saline (PBS) with ferrocene methanol added at a concentration of 1.2 mM as a redox mediator and in Fusayama/Meyer artificial saliva solution (7.1 pH) in accordance with the ASTM F2129 standard. Scanning electrochemical microscopy (SECM) was then used to determine the rate of oxide formation by preforming a series of approach curves at time intervals of 3 minutes for a total of 36 minutes for each sample. The approach curves did not readily fit to a system under kinetic control, which merits further study. The electrochemical results suggest that boron additions greater than 0.01 wt.% significantly slowed the oxidation rate. The electrochemical data indicated that the Ti2820 alloys displayed a higher corrosion potential (> -0.39 V vs. SCE) and higher corrosion current density (< 14.01 nA/cm2) as compared to the Ti64 control alloy. In conclusion, titanium-niobium-zirconium alloys are promising potential replacements for Ti64 dental implants due to their comparable corrosion properties, preferred mechanical properties and non-toxicity.
Support from the the LA section of NACE International, Western States Corrosion Seminar and the Western Area is acknowledged. The use of the SEM/EDS was made possible through an NSF MRI (DMR – 1429674) grant.
Poster #: 47
Campus: CSU Sacramento
Poster Category: Bioengineering
Keywords: artificial cells, actin, vesicles
Project Title: Modeling Cells With Giant Vesicles Encapsulating Polymerized Actin Networks
Author List:
Sanders, Nicholas; Undergraduate, Physics and Astronomy, California State University, Sacramento, Presenting Author
Purushothaman, Sowmya; Biomedical Engineering, University of California, Davis
Su, Wan-Chih; Biomedical Engineering, University of California, Davis
Parikh, Atul; Biomedical Engineering, University of California, Davis
Morris, Eliza; Faculty, Physics and Astronomy, California State University, Sacramento
Jensen, Mikkel; Faculty, Physics and Astronomy, California State University, Sacramento
Abstract: Cell mechanics govern a wide range of cellular function and plays a key role in cellular disease. For example, metastasizing cancer cells are softer than healthy cells, allowing them to migrate through narrow constrictions during metastasis. Artificial cell model systems provide a physical model to emulate living cells. Chemically triggered artificial cells have been extensively studied, but the mechanics of living cells have comparatively been studied less thoroughly. Most of the cell’s mechanical properties are governed by the cell’s biopolymer cytoskeleton, necessitating a good model system of artificial cells that incorporate cytoskeletal mechanical elements.
In this work, we successfully engineered a model artificial cell made of a polymerized actin network encapsulated in a lipid bilayer to form an artificial cell model system. Using confocal fluorescence and epifluorescence microscopy, we imaged and characterized polymerized actin encapsulated in a single-phase lipid vesicle. We were able to produce actin-containing vesicles with high yield, which remained stable over several hours. If vesicle bursting was induced by changing osmotic pressure, the actin network maintained its shape instead of diffusing quickly into the surrounding environment, demonstrating that the actin was polymerized and had successfully formed an entangled biopolymer gel. We also demonstrated that presence the actin network can stabilize osmotically induced pores for several minutes at a time as the network elasticity balances the line tension of the membrane pore. Together, our results suggest that a simple artificial cell system can capture many important features of living cells, and can be used as a model system to study cell mechanics.
Future studies will expand upon our current system by varying osmotic conditions, lipid membrane composition, and network crosslinking conditions. For example, we aim to control polymerization and depolymerization of the actin network by dialing up and down the osmotic pressure, which to our knowledge would demonstrate the first mechanically triggered response in an artificial cell system. These future tests would increase control of the model and improve its utility in modeling living cells.
Poster #: 48
Campus: CSU Fullerton
Poster Category: Bioengineering
Keywords: physical rehabilitation of post-stroke patients, mirror neuron, virtual reality
Project Title: Pilot Testing of Upper Limb Robot Rehabilitation System for Post-Stroke Patients
Author List:
Tang, Wesley; Undergraduate, Computer Engineering, California State University, Fullerton, Presenting Author
Chen, Binyun; Graduate, Mechanical Engineering, California State University, Fullerton, Presenting Author
Robson, Nina; Faculty, Mechanical engineering, California State University, Fullerton
Abstract: This research describes the initial testing of the ARWED, which is a virtual reality system for physical rehabilitation of patients with reduced upper extremity mobility resulting from a stroke. The purpose of the ARWED is to increase limb Active Range of Motion. The system performs a symmetric reflection of the patients’ healthy limb into a virtual 3D photorealistic model and maps it in real time on to their most affected limb, tapping into the mirror neuron system and facilitating the initial learning phase. Using the developed system, pilot experiments tested the extension of the action-observation priming effect linked to the mirror-neuron system on healthy subjects and one post-stroke patient. The initial assessment of the developed virtual photorealistic 3D hand models with healthy subjects imply that the developed models prime the human motor system in a manner consistent with the human model.
As a next step, the ARWED was tested on one post-stroke patient for the period of four weeks (one session/week). To assess the outcomes, the patient was tested on standard Box and Blocks and Fugl-Meyer tests prior and after the four-week ARWED therapy. Both tests are standard and assist in assessing the sensorimotor recovery after stroke. Movement time and Reaction time were measured through electromyography and 3D motion tracking system. The four-week ARWED testing showed that people with reduced joint motions can react to computer animations, link those animations onto joint motions, and learn to move successfully with a constraint. The preliminary assessment of the ARWED revealed that overall there are no changes in the assessment scores with only 4 rounds of device training per patient. However, the pilot electromyography tests showed that the virtual reality mirror therapy could trigger muscle activation in patients that are more than 3 years post-stroke. This can further serve as evidence that the time needed for recovery from stroke is not limited to one year and that additional practice can improve mobility in both the sub-acute and chronic phases following a stroke. It was concluded that patients may benefit from increased frequency and duration of training with the device to assess for any changes in the assessment scores. The future directions include clinical testing of the ARWED at UCSD Medical Center with increased frequency and duration of training with the device.
Poster #: 49
Campus: CSU Fullerton
Poster Category: Bioengineering
Keywords: natural physiological motion, canine orthotic wheelchair, parallel linkage design
Project Title: The Wearable Articulated Low-cost Canine (WALC) Wheelchair for Assisting Natural Physiological Motion
Author List:
Chen, Binyun; Graduate, Mechanical engineering, California State University, Fullerton, Presenting Author
Lopez, Myriam; Undergraduate, Mechanical Engineering, California State Polytechnic University, Pomona, Presenting Author
Nguyen, Kha; University of California, San Diego, Engineering
Robson, Simone; Fountain Valley High School
Gasser-Brenan, Aeden; North Hollywood High School
Abstract: This project is inspired by the four-legged chihuahua Heidi who suffers erosive immune polyarthritis, resulting in difficulty walking. Dog wheelchairs are expensive, heavy, do not have climbing abilities, or allow the dog’s body to tilt during sniffing and sitting. To alleviate these issues a novel type of low-cost, supporting orthotic wheelchair suitable for small-sized dogs is developed and tested.
To understand the key parameters that need to be taken into account, the first step was to study the biomechanics of canine motion. Motion capture data from a healthy dog walking at 0.55 m/s on a treadmill, sniffing, sitting and climbing curbs/high objects was obtained. The data analysis supported the overall idea that quadrupeds habitually power locomotion with their hindlimbs. The analysis also revealed that the dog mechanics of walking resembles a passive stiff-limbed planar parallel linkage model, which is in agreement with existing literature. Thus, the hypothesis was that developing a wearable wheelchair that incorporates articulated parallel linkages with two wheels on each side of the dog’s body will allow for a natural body motion and lead to increased comfort compared to the traditional orthotic devices. Each parallel linkage model consisted of a parallel linkage for the hindleg, a back with a distributed mass, and another parallel linkage for the front leg. A Solidworks CAD model of the wheelchair was designed based on the obtained motion capture data. To reduce the cost and the weight, the wheelchair was 3D printed and experimentally tested.
Test results and analysis showed that the walking trajectories of the dog using the wheelchair were within 2% standard deviation compared to the natural healthy trajectories enabling the desired repetitive physiological gait and curb climbing. The comparison between the sit and sniff natural trajectories with those of the wheelchair showed about 12.5% error. The authors believe that the latter was caused by some of the dog’s uncontrolled movements due to Heidi’s condition, resulting in a slight discomfort. It was concluded that, although some future motor learning might be needed in order for dogs suffering erosive immune paralysis to successfully control the sniff/sit patterns, the incorporation of planar parallel linkages for canines’ wheelchairs is sufficient for coordinating walking, sniffing and sitting motion and obtaining natural physiological gait.
Poster #: 50
Campus: Sonoma State University
Poster Category: Bioengineering
Keywords: Electrophysiology, Computational Neuroscience, Electrical Engineering
Project Title: Inference of willed attentional focus via local field potentials in humans.
Author List:
Bengson, Jesse; Faculty, Psychology, Sonoma State University
Holm, Adam; Undergraduate, Psychology, Sonoma State University, Presenting Author
Nadra, John; Undergraduate, Psychology, Sonoma State University, Presenting Author
Falk, Rachel; Undergraduate, Biology, Sonoma State University
Abstract: Studies of the cognitive neuroscience of visual-spatial attention have almost exclusively used instructional cues, often in the form of arrows, to direct attention to relevant locations. In everyday vision however, attention is often focused volitionally, in the absence of external signals. Although investigations of cued attention comprise thousands of neuroscientific studies, remarkably few studies of attention have addressed the challenging question of how spatial attention is initiated and controlled in the absence of external instructions, which we refer to as decision-driven attention. The isolation of a neural signature that reliably reflects an internal and intentional cognitive state such as attention is a crucial first step towards building a Brain Computer Interface in which individuals can reliably and robustly control devices by controlling internal cognitive states. We present data from a novel experimental paradigm in which individuals’ attentional focus can be inferred via the implementation of machine learning algorithm approach that measures frequency specific changes in the human brain’s local field potential as decisions to attend are implemented. Results indicate that the primary frequency-bands that differentially oscillate in response to binary internal decisions are in the 8-13 Hz (alpha) range recorded over the human visual cortex. This finding is crucial for the development of a reliable and generalizable brain-computer interface system that requires minimal training.
Poster #: 51
Campus: San Diego State University
Poster Category: Bioengineering
Keywords: Particle Image Velocimetry , Heart Failure, Intraventricular Flow
Project Title: Aortic insufficiency during Left Ventricle Assist Device support: a mock loop study
Author List:
Vu, Vi; Graduate, Mechanical Engineering, San Diego State University, Presenting Author
Balagtas, Camille; Young Scholar Program
Fujita-Willabus, Ana; Young Scholar Program
Lok, Megan; Young Scholar Program
May-Newman, Karen; Faculty, Mechanical Engineering, San Diego State University
Abstract: Aortic valve insufficiency (AI) is a serious complication in Left Ventricle Assist Device (LVAD) patients, which decreases cardiac output and systemic perfusion. Even initially normal aortic valves (AoV) have developed AI following long-term LVAD support, and has been associated with a significant reduction in patient survival. During LVAD support, the pressure difference across the AoV is increased, thus reducing AoV opening during systole and exacerbating regurgitant flow, which likely contributes to the long term pathology. The goal of this study was to develop a repeatable and reversible AI model in a mock circulatory loop, and study the effect of LVAD support on the clinical indices associated with worsening AI. A silicone model of a dilated left ventricle was attached to a rotary LVAD (Abbott Labs, IL) and tested for a Pre-LVAD baseline (BL) condition of 20% ejection fraction, and at three LVAD speeds. AI was created with a small 3-D printed stent that was non-obstructive to forward flow but prevented the AoV from fully closing. LV and aorta pressure, as well as LVAD and distal aorta flow were recorded at 200 Hz (LabChart, AD Instruments). Data were analyzed with LabChart and Excel. At BL, the mean aortic pressure was 622 mmHg with 2.3 L/min cardiac output. AI maintained a similar pressure but reduced flow to 1.8 L/min, producing 32% regurgitant fraction, which is considered mild-moderate AI. At 6400rpm LVAD support, the BL total systemic flow was 4.3 L/min, and 3.1 L/min for AI. The reduction of systemic flow accompanying by LVAD flow of 3.6 L/min indicates the presence of regurgitant loop in AI case. Net forward flow through the AoV decreased progressively with LVAD speed, with 23% of the total at 4800rpm, 12% at 5400rpm and 0% at 6400rpm. AI reduced forward flow and increased backward flow that progressed with LVAD speed, such that the net forward flow was 0% at 4800rpm and -15% at 6400rpm. This reduction resulted from a combination of decreased forward flow through the AoV during systole, and increased backflow during diastole. A small but consistent increase in the transvalvular pressure accompanied the worsening AI. Overall, the results showed that an initially mild level of AI worsened with LVAD support prior to any remodeling, simply due to the altered biomechanics of the AoV. These findings provide a foundation for new LVAD control strategies that can restore the AoV biomechanics and reduce the impact of AI on patients with LVAD support.
Poster #: 52
Campus: CSU Long Beach
Poster Category: Bioengineering
Keywords: Tissue Engineering, Cardiac Fibrosis, Fibroblasts
Project Title: Bioengineered 3D Fibrosis Model
Author List:
Suarez, Jessica; Undergraduate, Biomedical Engineering Department, California State University, Long Beach, Presenting Author
Mata, Daniel; Undergraduate, Biomedical Engineering Department, California State University, Long Beach, Presenting Author
Kane, Madison; Undergraduate, Biomedical Engineering Department, California State University, Long Beach
Hoang, Thuy ; Undergraduate, Biomedical Engineering Department, California State University, Long Beach
Ayala, Perla; Faculty, Biomedical Engineering Department, California State University, Long Beach
Abstract: Heart failure affects 5.7 million adults in the United States. Cardiac fibrosis is a leading cause of heart failure after a myocardial infarction (MI). Following an MI, cardiac fibroblast cells respond by rushing to the injured site and differentiating into myofibroblast cells. Excess proliferation of myofibroblasts forms a scar tissue surrounding the injured site. The purpose of this research is to investigate methods to modulate fibroblast proliferation and myofibroblast activation in an in vitro 3D model. We engineered a 3D tissue environment to investigate modulation of fibroblasts on different topographical environments. A 3D hydrogel from extracted porcine collagen type I was prepared within 3D poly-dimethyl siloxane (PDMS) molds with and without micro-channels. Fibroblasts were cultured at different time points (1, 3, and 5 days) within the engineered 3D environment. Cells were stained using Calcein AM and immunostaining of alpha-actin. MTT Assay was used to correlate cell density. Microscope imaging showed that cells within the micro-channels have a more defined structure than the cells in the flat molds. Preliminary results suggest that fibroblast activation and proliferation are influenced by the microtopographical environment. The results of this analysis of 3D constructs can potentially be applied to minimizing not only cardiac fibrosis, but all forms of fibrosis throughout the human body.
ACKNOWLEDGEMENTS:
This research was supported by CSUPERB, CSULB ORSP, CSULB COE SFG, LSAMP, and BUILD (National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers; 5UL1GM118979; 5TL4GM118980; 5RL5GM118978). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Poster #: 53
Campus: Cal Poly Pomona
Poster Category: Bioengineering
Keywords: MEMS, Biocompatibility, Sensors
Project Title: Fabrication and Multiphysics Model of MEMS Thermal Flow Sensor
Author List:
Cable, Justin; Graduate, Mechanical Engineering, California State Polytechnic University, Pomona, Presenting Author
Anderson, Kevin ; Faculty, Mechanical Engineering, California State Polytechnic University, Pomona, Presenting Author
Abstract: The miniaturization, biocompatibility, sensitivity and versatility achieved with thermal flow sensors has drawn interest in multiple engineering disciplines. Multiple configurations of a micromachined thermal flow sensors are presented demonstrating a liquid flow rate on the scale of microliters per minute. Such flow sensors are capable of being used in various biomedical applications. For example, thermal flow sensors can be implemented in implantable micro-pumps to deliver drugs without the need for inserting a needle in the human skin. These flow sensors rely on the varying electrical signals of sensors created by the heat transfer generated by different flow rates. The sensor arrays presented were constructed using microelectromechanical systems (MEMS) techniques including micromolding, wet etching and dry etching utilizing USP class VI biocompatible materials. A numerical approach via COMSOL was used to predict and describe the electrical, thermal and fluid behavior of the sensor which was later verified with experimental data. The array of sensors was used to characterize the heat distribution created by a resistive heater and compared to the simulations and existing analytical solutions. This method is known as the calorimetric sensing principle and is one of three major thermal flow sensing methods. The construction allowed for multiple thermal flow sensing operational modes including hot-film, calorimetric, and time-of-flight without changing the design. The calorimetric sensing principle was shown to have superior sensing capabilities similar to previously reported MEMS thermal flow sensors. For this reason, the heat distribution around a centralized heater between several temperature sensors was more thoroughly investigated. The variety of flow sensor geometries constructed were compared to investigate maximum heat transfer to the sensors, thermal insulation, size, sensitivity and range capabilities. Performance characterization for the sensor used measurement uncertainty theory to reflect the probability distribution of measured flow rate data as they relate to the actual values. The sensor design is such that it is capable of detecting different flow directions and various flow ranges for different fluids. In addition, to the performance capabilities outlined, the sensor is relatively inexpensive, should have a long lifetime due to the lack of moving parts, and can be integrated into an existing circuit board for easy electrical connections.
Poster #: 54
Campus: Cal Poly Pomona
Poster Category: Bioengineering
Keywords: microbiological, corrosion, biofilm
Project Title: Microbiologically Influenced Corrosion in Marine Environments – Surface Roughness Effects
Author List:
Sena, Grazziela; Graduate, Chemical and Materials Engineering, California State Polytechnic University, Pomona, Presenting Author
Patel, Neelam; Undergraduate, Chemical and Materials Engineering, California State Polytechnic University, Pomona, Presenting Author
Fly, John; Undergraduate, Biological Sciences, California State Polytechnic University, Pomona
Ravi, Vilupanur; Faculty, Chemical and Materials Engineering, California State Polytechnic University, Pomona
Abstract: Microorganisms can influence the corrosion rate of metallic materials through a phenomenon known as microbiologically influenced corrosion (MIC). MIC can increase corrosion rates of metallic alloys utilized in corrosive environments, e.g., salt water corrosion experienced in marine infrastructures, thereby reducing the service life of these alloys. The objective of the current study was to determine whether the surface roughness of an alloy has a significant effect on its corrosion resistance in the presence of microorganisms. Three test coupons each (N = 3) of marine grade carbon steels and stainless steels (UNS G10180, UNS S30400 and UNS S31603) were ground to multiple finishes (80 grit, 600 grit and 0.05 µm) to achieve various surface roughnesses. The cylindrical test coupons, each 6.0 ± 0.2 mm long and with a diameter of 13 ± 0.1 mm, were immersed in biologically active seawater collected off the coast of Southern California. The mass differences between pre-and post-corroded coupons were recorded and the corrosion rates were determined using an equation per the ASTM G102 standard. Post-test coupons were examined using optical and scanning electron microscopy coupled with energy dispersive spectroscopy (SEM/EDS).
Corrosion rates were calculated for each of the alloy coupons for 24 and 48 hour exposures as well as 7 and 14 day exposures in natural seawater. The overall trend was that corrosion rates decreased with increasing exposure time. For example, UNS31603 coupons with an 80 grit surface finish had average corrosion rates of 135 ± 6 µm per year for 7 days and 62 ± 3 µm per year at 14 days relative to the 24 and 48 hours test coupons, i.e., 1011 ± 51 µm per year for 24 hours and 589 ± 30 µm per year for 48 hours. For UNS S30400 and UNS S31603 coupons, this decrease in the corrosion rates with increasing exposure times can be attributed to the formation of a protective passive film. However, the same trend was seen in the UNS G10180, a material that does not form a protective oxide film. One possible explanation for the latter may be the formation of a biofilm inhibiting further corrosion. The surface roughness effects were more nuanced with different trends being exhibited by the different alloys.
Support from the Southern California Marine Institute, the LA section of NACE International, Western States Corrosion Seminar and the Western Area is acknowledged. The use of the SEM/EDS was made possible through an NSF MRI (DMR – 1429674) grant.
Poster #: 55
Campus: CSU Long Beach
Poster Category: Clinical
Keywords: depression, side-effects, addiction
Project Title: Functional consequences of juvenile ketamine administration on the rewarding effects of ethanol in male and female adolescent rats.
Author List:
Franco, Daniela; Graduate, Psychology, California State University, Long Beach, Presenting Author
Zamudio, Jennifer; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Iniguez, Sergio; University of Texas, El Paso
Zavala, Arturo; Faculty, Psychology, California State University, Long Beach
Abstract: Although Major Depressive Disorder (MDD) is the leading cause of disability worldwide, current antidepressants continuously fail to treat depressive symptoms in more than half of all cases of depression. Recently, ketamine has demonstrated rapid and effective antidepressant effects in clinical and preclinical models. However, since ketamine is a drug of abuse, it may harbor potential long-term effects in juvenile populations. Specifically, ketamine may inadvertently alter the developing brain and increase one’s vulnerability to other drugs of abuse, such as alcohol. Alcohol is the most commonly abused recreational drug in the United States. Given that alcohol use typically begins in adolescence, we investigated whether early-life ketamine administration increases the rewarding effects of ethanol, the type of alcohol that is consumable, in adolescence. We hypothesized that early administration of ketamine enhances the preference for ethanol, as measured using the conditioned place preference (CPP) model, a validated animal model of drug reward. Male and female Sprague-Dawley rats were treated daily with ketamine from postnatal day (PD) 21-30, a period of rat development comparable to late childhood and the start of adolescence. Rats were then assessed for ethanol-induced CPP beginning on PD 31 using a 10-day CPP procedure. During days 1 and 10 of the CPP procedure, rats were tested for their preconditioning and postconditioning place preference, respectively, in 20-minute sessions. During days 2-9, rats were conditioned 15-minutes a day with either ethanol (0, 0.125, 0.5, or 2.0 g/kg) or saline on alternating days. Results demonstrate that periadolescent exposure to ketamine reverses the adverse effects of 2.0 g/kg of ethanol, as male rats pretreated with saline exhibit a strong aversion to the drug-paired side, while ketamine pretreated animals continue to show a preference for the ethanol-paired side. In females, early-life ketamine administration did not alter the preference for ethanol. These results suggest ketamine pretreatment reverses the observed aversive properties of ethanol, likely increasing the abuse potential of ethanol. Overall, these results suggest early-life ketamine administration may have long-lasting changes in the rewarding effects of alcohol, particularly in males, and that the use of ketamine as an antidepressant may have potential detrimental side effects.
Poster #: 56
Campus: CSU Long Beach
Poster Category: Clinical
Keywords: wearable technology, bedsores, pressure ulcers
Project Title: Smart Unit for Bedsore Prevention (Smart-UP): Utilizing a Smart Wearable Technology to Prevent and Manage Bedsores
Author List:
Soangra, Rahul; Crean College of Health and Behavioral Sciences, Chapman University, Irvine, CA, Presenting Author
Krishnan, Vennila; Faculty, Physical Therapy, California State University, Long Beach
Abstract: Bedsores affect about 2.5 million people in the US and costs about $9.1-$11.6 billion per year. Bedsores are skin wounds that result from prolonged pressure on the skin that is in contact with a bed or wheelchair. Bedsores or pressure injuries are painful, take a long time to heal and are often a precursor of life-threatening complications leading to morbidity and mortality in older adults. Orange County alone is home to over 3.1 million people and is the sixth most populous county in the US. It is expected that 25% of this population will be above the age of 65 years by 2040. It is important for us to be able to respond to the health concerns of this rapidly growing population of older adults including the need to heal and prevent bedsores. We developed a wearable technology based smart device for bedsore prevention and management. In this feasibility study, seven healthy younger adults (26.7 ±4.2 years) and seven older adults (73.7 ± 7.9 years) participated and this study was approved by Chapman University IRB. The sensor was positioned at L5/S1, above posterior iliac spine and all activities of daily living ranging from standing /sitting to night-time sleeping were collected for 3-days continuously. We found that older adults performed significantly higher low amplitude movements when compared to younger adults (p<0.05). We also found younger adults carried out significantly more high frequency movements compared to older adults (p<0.05). Our results also show that older adults make significantly less transitions per hour of sleep during the nighttime (p<0.05). The goal of this work was to investigate novel intervention methods to understand activities of daily living in young and older adults using wearable sensors. This information is the first step to understand movement patterns in older adults and is critical in developing preventing measures and managing bedsores in this population.
Poster #: 57
Campus: San Francisco State University
Poster Category: Clinical
Keywords: tele-rehabilitation, mHealth, stroke
Project Title: An evaluation of an upper limb stroke rehabilitation system for medically underserved populations
Author List:
Hughes, Charmayne; Faculty, Health Equity Institute, San Francisco State University, Presenting Author
Mariscal, Tatiana; Graduate, Kinesiology, San Francisco State University, Presenting Author
Sera, Michael; Graduate, Interdisciplinary Studies: Bioengineering and Rehabilitation Technologies, San Francisco State University
Cruz, Zeus; Undergraduate, Electrical Engineering, San Francisco State University
Warner, Sam; Undergraduate, Kinesiology, San Francisco State University
Hintze, Amy; San Francisco State University
Rashidi, Neela; Undergraduate, Kinesiology, San Francisco State University
Abstract: Each year approximately 15 million people worldwide suffer a stroke, and five million of those are left with some type of permanent upper limb disability that has a detrimental effect on patients’ capacity for independent living and economic self-sufficiency. Conventional stroke therapy is a labor intensive process, which involves daily one-on-one interactions with therapists that can last for several months. This mode of rehabilitation places a significant burden on physical therapists and clinics in medically underserved areas and populations that struggle with a shortage of health providers and technical resources crucial to the delivery of physical rehabilitation services. In an attempt to improve stroke rehabilitation outcomes in medically underserved areas and populations, we have developed the outREACH system for the delivery of personalized upper extremity stroke assessment and rehabilitation in an individual’s home environment. In this study, we utilized a user-centered design approach to evaluate the usability of the outREACH system for the delivery of personalized upper limb stroke assessment and rehabilitation in medically underserved areas and populations. We conducted an iterative usability study based on a mixed-methods approach, using semi-structured cognitive interviews, as well as a demographic and user experience survey. Three iterative usability testing cycles were completed; two from U.S. populations and one from Ethiopia. Thematic analysis and descriptive statistics were used to analyze the think-aloud tasks, interviews, and questionnaires. Two main usability issues in round 1 were identified with the scheduler module and the similarities between the Home screen and the My Care Plan screen. After applying modifications for the second iteration, there was a significant increase in the average usability score (from 43 to 88%), and a reduction in both the number of errors, as well as the time taken to complete each task. The results of the present study will be integrated into the development, integration, and evaluation of the outREACH system that supports stroke tele-rehabilitation in underserved populations.
Poster #: 58
Campus: CSU Fullerton
Poster Category: Clinical
Keywords: antibiotic resistance, ionophore, acinetobacter
Project Title: 8-Hydroxyquinoline derivaAuthor List:
Magallon, Jesus; Graduate, Biological Science, California State University, Fullerton, Presenting Author, Eden Award Nominee
Chiem, Kevin; Graduate, Biological Science, California State University, Fullerton
Ramirez, María S.; Faculty, Biological Science, California State University, Fullerton
Tolmasky, Marcelo; Faculty, Biological Science, California State University, Fullerton
Abstract: The increase in multidrug resistant bacteria seriously compromises our ability
to treat infections and complicates medical procedures that require preventive
treatment. Aminoglycosides are antimicrobials widely used to treat gram-negative
infections and amikacin is one of the most resilient against development of
resistance. However, the rise and dissemination of the aminoglycoside 6′-Nacetyltransferase
type Ib enzyme [AAC(6’)-Ib] has limited its use. AAC(6’)-Ib, an
aminoglycoside modifying enzyme found in numerous clinical isolates, mediates
inactivation of the aminoglycoside by acetylation. Its dissemination is leading to a
steady increase in resistant infections that results in an elevated number of
fatalities. We have recently found that low concentrations of Zn+2 reverses
amikacin resistance when combined with clioquinol (5-chloro-7-iodo-8-quinolinol),
an ionophore that may also be active in treatment of that is being explored as
treatment for cancer and neurodegenerative conditions. In this work we
compared the efficiency of clioquinol and other 8-hydroxyquinoline derivatives in
complex with Zn+2, to inhibit growth of amikacin resistant A. baumannii clinical
isolates in the presence of the antibiotic. Growth inhibition assays were carried
out in Mueller-Hinton broth with the addition of amikacin and the complexes in
microtiter plates using BioTek Synergy 5 microplate reader. Cultures were
carried out for 20 h at 37°C measuring OD600 every 20 minutes to assess growth.
A. baumannii A155 and A144 are clinical isolates that harbor aac(6’)-Ib and grow
in the presence of 8 μg/ml amikacin. Control cultures in the presence of
incremental concentrations of ZnCl2 or the ionophores showed that they did not
impair growth at the levels used in this work. The growth of A. baumannii A155
and A144 was completely inhibited in the presence of 8 μg/ml amikacin when 5
μM clioquinol or 5,7-diiodo-8-quinolinol and 25 μM Zn+2 were added to the growth
medium. Instead, all other derivatives tested showed little or no effect. Our
results show that the combination of ionophores and Zn+2 may be a viable
strategy to overcome resistance to amikacin and other aminoglycosides.
Prolonging the usefulness of existing antibiotics will preserve lives, save
resources by reducing the cost of treatment, and permit us to reserve the newest
antibiotics for exceptional situations.
Funding: Public Health Service grant 2R15AI047115 from NIAID, NIH and
CSUPERB.
Poster #: 59
Campus: CSU San Marcos
Poster Category: Clinical
Keywords: T cells, Chemokines, Luminex
Project Title: Regulation of Epidermal T Cell Function by C-C Chemokine Receptor 6
Author List:
Bshara-Corson, Savannah; Graduate, Biology, California State University San Marcos, Presenting Author
Miranda, Nadia; Undergraduate, Biology, California State University San Marcos, Presenting Author
Jameson, Julie; Faculty, Biology, California State University San Marcos
Abstract: The CDC reports that obesity affects 39.8% of the United States population. Patients who suffer from obesity and type 2 diabetes experience immunological dysfunction in the skin, where chronic inflammation can lead to non-healing wounds. Epidermal T cells play key roles in maintaining skin homeostasis by secreting cytokines and chemokines to facilitate inflammatory responses following tissue damage. During obesity and type 2 diabetes, epidermal T cells exhibit reduced expression of select cytokine and chemokine receptors including C-C Chemokine receptor 6 (CCR6). The major ligand for CCR6 is C-C Chemokine Ligand 20 (CCL20). Upon CCL20 binding, CCR6 induces lymphocyte migration. In this study, we investigated the role of CCR6 in regulating epidermal T cell function in the epidermis. Murine epidermal T cells were harvested and stimulated in the presence or absence of CCL20 to determine how CCL20 regulates epidermal T cells cytokine and chemokine expression. We found that CCL20 upregulates IL-2R on epidermal T cells and downregulates T cell receptor which are both signs of epidermal T cell activation. In addition, CCL20 costimulates TCR stimulation to activate epidermal T cells. Using flow cytometry and Luminex analysis we discovered that upon activation with anti-CD3 and CCL20 stimulation, epidermal T cell production
of select cytokine and chemokines increases. Furthermore, we suspect that CCR6 has a pivotal role in activation causing morphological changes to skin resident T cells. Future studies will determine how CCL20 regulates cytokine and chemokine expression on murine
models with type 2 diabetes. Understanding how CCR6 mediates cellular responses can lead to therapeutics for type 2 diabetic patients with chronic non-healing wounds.
Poster #: 60
Campus: CSU Monterey Bay
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: mathematical modeling, small data, tumor growth
Project Title: Fitting Classical Mathematical Models to Small Data Sets from Lewis Lung and Human Breast Carcinomas
Author List:
Hernandez, Maria Teresa; Undergraduate, Mathematics and Statistics, California State University, Monterey Bay, Presenting Author
Abstract: Classical mathematical models are useful in modeling tumor volume growth to predict future growth and evaluate treatment plans. However, it is difficult to measure regular and long-term growth in a clinical or lab setting for ethical and practical reasons. Therefore, we must develop methods to fit models to small data sets to develop models for clinical use. We explored the minimum number of observations necessary to confidently fit several common classical ordinary differential equation models (e.g., generalized logistic growth models) for the prediction of tumor growth via computer simulations in R. We simulated data using estimated parameters from previous in vivo experiments with mice that observed the growth of Lewis lung and human breast carcinomas, adding a biologically reasonable level of noise, and setting our time frame to one observation per day for 30 days. A notable distinction between the cancer types is that lung cancer has a faster growth rate. We used the deSolve package in R to fit ordinary differential equations models to the simulated data. After convergence of the solution resulted in optimized parameter estimates, we visually assessed the final model for fit to the observed data over specified time intervals and performance in the prediction of future tumor growth observations. The results of our analysis showed that the predictive accuracy limit and best fit limit for data generated from the logistic growth model is 25 days for breast cancer and 15 days for lung cancer. Similarly, for data generated from the generalized logistic growth model, the predictive accuracy limit and best fit limit was 20 days for breast cancer and 10 days for lung cancer, demonstrating that faster growing carcinomas may require less data to fit a model. Simulations in R allowed us to observe tumor growth for long periods without having to consider the ethical treatment of real in vivo test subjects. In the future, we will conduct simulations that more closely mirror real in vivo experiments to determine the best methods to fit models in real lab situations. This includes adding varying levels of noise, using different time frames, and using more sophisticated model selection methods, such as AIC, make model selection for fit and predictive tumor growth accuracy more concrete than just a visual inspection. Our goal is for our simulation experiment based on in vivo experiments to be extended to develop personalized treatment plans for cancer patients.
Poster #: 61
Campus: CSU Los Angeles
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: regression analysis, energy expenditure, EMG sensing
Project Title: Improvement in Accuracy of Regression Estimates of VO2 by From Wireless Sensors by Incorporating Muscle (EMG) Sensor Data
Author List:
Enciso, James; Graduate, Electrical Engineering, California State University, Los Angeles, Presenting Author
Bowser, Isaac; External Partner
Velasco, James; Undergraduate, California State University, Los Angeles
Martinez, Manuel G; Undergraduate, California State University, Los Angeles, Presenting Author
Vazques, Fabiola; Undergrauate, California State University, Los Angeles
Pebdani, Roxanna; University of Sydney
DeLeon, Ray D; Faculty, California State University, Los Angeles
Dy, Christine; Faculty, California State University, Los Angeles
Keslacy, Stefan; Faculty, California State University, Los Angeles
Won, Deborah; Faculty, California State University, Los Angeles
Abstract: Introduction
Tracking physical activity and energy expenditure can be conveniently performed using fitness trackers. However, most fitness trackers utilize metrics based on heart rate and accelerometry, which are subject to motion artifact noise – a limitation for tracking subjects performing wheelchair-based exercises, such as those with spinal cord injury. We are currently developing a mobile fitness system intended for individuals who perform exercises on wheelchairs to reliably estimate energy expenditure from body-worn, wireless sensors. We tested 3 regression-based methods to predict VO2 (an accepted metric of energy expenditure) from heart rate and muscle activity (EMG) to determine whether EMG provided any additional information and how much of a contribution it made over heart rate alone.
Materials and Methods
Six subjects – four with spinal cord injury who regularly used wheelchairs, and two non-injured non-wheelchair users – performed 2 sets of 3 different exercises in a wheelchair while the following data was simultaneously collected: oxygen consumption (VO2, Cosmed Metabolic Cart), heart rate (Wahoo Tickr), and muscle activation (EMG) (Dynofit Flexdot). EMG data was recorded from anterior and posterior deltoids, biceps, and triceps. Three regression-based methods for estimating VO2 were tested using:1) single linear regression using heart rate; 2) multiple linear regression using heart rate and all four EMG signals; and 3) logistic regression on heart rate.
Results and Discussion
VO2 estimates obtained from a single linear regression performed best when using heart rate compared to any individual EMG signal, although estimates using deltoid EMG data often reached similar R2 values depending on the exercise performed. The logistic regression yielded slightly higher R2 values than the single linear regression by an average of 18%, particularly in resistance exercises.
The multiple linear regression performed significantly better with goodness of fit values 26% higher on average over single linear regression. These improvements indicate that EMG data appears to contain information about energy expenditure that is non-redundant to heart rate. Further examination of the data shows that the additional information provided in EMG signals are changes that occur on a faster time scale relative to slower changes in heart rate. Thus, a more accurate fitness metric for a mobile fitness system is feasible through the incorporation of wireless EMG sensors.
Poster #: 62
Campus: Sonoma State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: breath disease detection, diabetes, wearable medical device
Project Title: Smart Wristband with Integrated Chemical Sensors for Detecting Glucose Levels using Breath Volatile Organic Compounds
Author List:
Harold, Casey; Undergraduate, Engineering Science, Sonoma State University, Presenting Author
Boubin, Matthew; Miami University
Shrestha, Sudhir; Faculty, Engineering Science, Sonoma State University, Presenting Author
Abstract: Volatile organic compounds (VOCs) in human breath have been known to carry markers of numerous diseases, including for hyperglycemia. It is projected that by 2030, 15.3% of Americans will have diabetes and that treatment of diabetes will cost the US economy a total of over 600 Billion US dollars in a year. With the prevalence of diabetes increasing at such an alarming rate, the presented smart wristband provides a non-invasive tool for monitoring blood-glucose (BG) levels. This poster presents our effort towards developing embedded system-based wearable solutions to estimate BG levels from VOCs in a patient’s breath. This work has two-fold initiative. First, three highly sensitive chemical sensors were tested in a controlled setup with artificial breath that mimics known VOC footprints of high and low BG levels. Using the data, a machine learning algorithm, support vector machine (SVM), was trained, and subsequently, implemented in a microcontroller. In real time-time testing using the artificial breath, the trained algorithm in the microcontroller classified low and high BG footprints in artificial breath samples with a high accuracy of 97.07%. The implementation of SVM in a microcontroller is significant as it enables development of portable and wearable BG monitoring devices. Secondly, a smart wristband prototype that integrates the three chemical sensors tested above, was developed. An Arduino-based wearable microcontroller platform was used for its small form factor and a low-power operation. A case for the wristband was created using a 3D printer. The wristband, powered by an on-board 3.7 V 500 mAh rechargeable Li-ion battery, reads the sensors and sends the data to a smartphone. A Bluetooth module in the wristband enables communication with the smart phone. To conserve power, when not in use, the wristband enters into a low-power consumption mode. A vendor provided smartphone app was used for communicating with the wristband, collecting sensor data, and uploading the data in the cloud. Ongoing efforts in this work includes implementing the SVM in the wristband microcontroller and conducting tests with artificial breath. The presented work makes a significant contributions toward the development of wearable devices for detecting BG levels from a patient’s breath. It also has a broader implication in enabling passive-continuous health monitoring solutions and assisting in diagnosing diseases that are hard to catch through current diagnostic practices.
Poster #: 63
Campus: CSU Fullerton
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: RNA-seq, adenovirus, glial cells
Project Title: Transcriptomic analysis of PAX5-induced MCOLN2 expression reveals a potential approach to restrict reactive gliosis in Mucolipidosis IV brain
Author List:
Chacon, Jonathan; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Newara, Kelly; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Rosas, Lauren; California State University Fullerton
Cuajungco, Math; Faculty, Biological Science, California State University, Fullerton
Abstract: Mucolipidosis type IV (MLIV) is a lysosomal storage disorder caused by mutations or deletions in the Mucolipin-1 (MCOLN1) gene, encoding the transient receptor potential Mucolipin-1 (TRPML1) protein. TRPML1 belongs to a subfamily of cation channels that includes TRPML2 and -3, encoded by MCOLN2, and -3 genes, respectively. MLIV is characterized by cognitive and psycho-motor issues and affects mostly children and young adults of Ashkenazi Jewish descent. Recently, it was shown that the cerebral cortex and cerebellum of Mcoln1 knockout mice exhibit extensive gliosis, which is consistent with inflammatory response. It is not clear, however, whether inflammation is the cause or consequence of MLIV. Due to a high degree of sequence homology, we propose that TRPML2 could substitute for the loss of functional TRPML1 in MLIV, potentially rescuing the disease. As a proof-of-principle, we used MCOLN2’s transcription factor called PAX5 to induce its expression in human neuroglioma cells using adenovirus. We found that PAX5 up-regulated endogenous MCOLN2 transcripts as evidenced by standard polymerase chain reaction (PCR) and real-time quantitative PCR. To study the effects of PAX5-induced upregulation of MCOLN2 transcripts on global gene expression, we collected total RNA from treated and control cells for subsequent paired-end RNA sequencing (RNA-seq). We performed transcriptome analysis using both commercial and open-source software. Enrichment analysis of RNA-seq data revealed marked reductions in gene ontology (GO) terms pertaining to cytokine production and innate/adaptive immune responses in PAX5-treated cells compared to negative controls. Experimental validation of the GO list showing down-regulated expression due to PAX5-induced MCOLN2 expression is currently being done using real-time quantitative PCR. Given the growing evidence of early gliosis from aberrant pro-inflammatory cytokine production as contributing factor to MLIV disease pathogenesis or progression, our results indicate that forced induction of endogenous MCOLN2 expression in human glial cells may potentially ameliorate some of the pathologic changes seen in the disease. Future studies on forced induction of Mcoln2 in MLIV mouse brain could open new research avenues to determine whether Trpml2 could dampen down gliosis and possibly prevent neurodegeneration in MLIV patients.
Poster #: 64
Campus: CSU San Marcos
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Population Genetics, Type 1 Errors, FDR Corrections
Project Title: Inconsistencies in Type 1 Error Corrective Methods used in Population Genetics Studies
Author List:
Gonzalez, Nicolette; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Mey, Ken; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Kansagra, Khyati; Undergraduate, Biological Sciences, California State University San Marcos
Grenier, Christy; Undergraduate, Biological Sciences, California State University San Marcos
Nunez-Zavala, Stefany; Undergraduate, Biological Sciences, California State University San Marcos
Summerhays, Bryce; Graduate, Biological Sciences, California State University San Marcos
Wulf, Gwendalyn; Undergraduate, Biological Sciences, California State University San Marcos
Sethuraman, Arun; Faculty, Biological Sciences, California State University San Marcos
Abstract: Population Genetic studies commonly use multiple independent tests when making broad “family-wise” inferences about the current disposition of a population, and look to measure qualities such as: Hardy-Weinberg Equilibrium (HWE), Linkage Disequilibrium (LD), Population Differentiation (ie. Fst, Gst), etc. However, when performing statistical tests across multiple independent pairs of genomic loci (or populations), the false positive rate grows at the Family-Wise Error Rate (FWER) of 1-(1-alpha)^k, where alpha is the set threshold for the significance level. This significance level is often set to a standardized value of alpha=0.05, or a “less stringent” threshold value, which results in an inflated False Discovery Rate (FDR) and FWER’s, when comparing multiple loci. Correspondingly, the true positive rate across tests declines. Correction methods such as: Bonferroni, sequential Bonferroni, Benjamini and Hochberg (B-H method), and Benjamini and Yekutieli (B-Y method) are commonly utilized to lessen inflation of FDR, but vary on their level of stringency when setting the threshold value for statistical significance. We searched thousands of recent population genetics literature (2013-present) and retained 205 papers which assessed all three population attributes of interest: tests of HWE, LD, and Fst, in order to test whether inconsistencies were present in the multiple testing correction methods used (if any). A Fisher’s exact test of independence in correction methods used was performed between tests of HWE and differentiation and tests of LD and differentiation, and indicated inconsistent usage of correction methods (p-value < 0.0001). A Chi-squared goodness of fit test was performed (chi2 = 40.85, p-value = 5.2e-6) which indicated that less stringent corrections methods were largely being utilized over more stringent methods for retaining more “interesting” loci that were considered as Fst outliers, further perpetuating inconsistencies in the use of correction methods. We show conclusively that editors and reviewers should necessitate authors to communicate their use of correction methods applied, and become aware of whether they are consciously, or unconsciously avoiding consistently applying them.
Poster #: 65
Campus: Sonoma State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: computed tomography, convolutional neural network, interstitial lung disease
Project Title: A computational framework based on convolutional neural network for classifying interstitial lung disease in computed tomography scans
Author List:
Havranek, Christian; Undergraduate, Computer Science, Sonoma State University, Presenting Author
Penna, Skyler; Undergraduate, Computer Science, Sonoma State University, Presenting Author
Gill, Gurman; Faculty, Computer Science, Sonoma State University
Abstract: Convolutional Neural Networks (CNN) have widely become the method of choice for computer aided detection of interstitial lung diseases (ILD) in computed tomography (CT) scans. ILD is a group of diseases that cause progressive scarring on lung tissue and manifest themselves with distinct patterns in CT. CNNs consist of layers that are trained to learn such patterns and automatically detect the diseases, which assists the doctors in speeding up the diagnosis time. However, CNNs need a large amount of data (in million of images) to be trained, which is not typically available for medical applications. A database widely used for ILD classification, MedGIFT database, contains CT scans from only 128 patients diagnosed with one or more of 10 different ILDs or as healthy. This research developed a CNN based computational framework to classify images when the training set is small and applied it for classifying image patches extracted from the MedGIFT database. A method called transfer learning was incorporated wherein the CNN is initially pre-trained with a database of a million natural images and the learned network is ‘transferred’ to the medical images by appending and training a single additional CNN layer. This layer can be appended after any layer in the original CNN and classification accuracy can be measured.
The CNN architecture used in this research is Inception-v3 (Google LLC) pre-trained with ImageNet database. Inception-v3 requires its input to be three channel images, so a framework was developed to extract patches in CT slices from within region of interests (ROI) marked with a specific disease label. Patches were converted from having Hounsfield Unit (HU) values, a pixel value designating density, into corresponding 8-bit Red, Green, and Blue (RGB) channels. The framework allows for user-defined patch sizes, frequency of patch extraction, and HU to RGB conversion ranges.
Multi-class classification (categorizing a patch as emphysema, fibrosis, ground glass, micronodules or healthy) tests were ran with 5669 patches gathered by this framework. The optimal location for appending additional layer proved to be one of the earlier layers which yielded an average F1 score of 83.66 +/- 4.75%. Tools like data augmentation (artificially increasing number of images), ensembles (combining results of several CNNs), and fine tuning (training entire network) have been developed as part of the computational framework and will be applied to further improve results.
Poster #: 66
Campus: CSU Long Beach
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Diabetes, Modeling, artificial pancreas
Project Title: Modeling the Blood Glucose Concentration for Patients with Type I Diabetes
Author List:
Perez, Anthony; Graduate, Department of Chemical Engineering, California State University, Long Beach, Presenting Author
Kelly, Jasper; Graduate, Department of Chemical Engineering, California State University, Long Beach, Presenting Author
Yang, Yu; Faculty, Department of Chemical Engineering, California State University, Long Beach
Abstract: The type I diabetes is life-threatening and leads to many complications. About 1.25 million Americans have type 1 diabetes and this number grows by 40,000 per year. To keep patients alive and ensure their life quality, an artificial pancreas is the most crucial device. It is a control system to regulate blood glucose concentration (BGC) through automatic insulin injections. Due to the individual differences, it is of importance to build a personal and simple BGC model to predict the dynamical response of the glucose to the insulin injection. This model will enable the artificial pancreas to determine the optimal insulin amount and correct time of injection for individual patient. To build such model, we use an FDA-accepted in-silico simulator to conduct the multi-step test and collect the glucose response data for patients at different ages. Then four types of empirical models, including state space, autoregressive with exogenous terms (ARX) model, Box-Jenkins (BJ) model, and nonlinear ARX, are constructed by fitting the measurement data. The state space model can be identified through the subspace method, which requires less engineering efforts and online computations. The ARX model can be identified through the least squares and is friendly to controller design. The BJ model can be identified through the prediction error method, which uses previous prediction error to correct the future forecast and thus has better accuracy than other linear models. Nonlinear ARX model based on the artificial neural networks (ANN) is more complex but able to approximate nonlinear dynamics of the BGC without using any prior knowledge. This project aims to make a comprehensive comparison study on these models and determine the best model for the controller design in an artificial pancreas. Our results show that the nonlinear ARX model is the best predictor for BGC (above 80% fitness), but it requires significant online computations due to the complex ANN. Moreover, the overfitting may happen if the parameters of ANN are not well-tuned. On the contrary, a high-order ARX model, even though is slightly less accurate (70~75% fitness), is more suitable for control system design because of its simple structure with much fewer number of parameters than other models. This research establishes the basic model used in the artificial pancreas and our next step is to develop a model predictive controller of the BGC based on this model.
Poster #: 67
Campus: Cal Poly San Luis Obispo
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: tissues-on-a-chip, interstitial flow, microfluidics
Project Title: Variable-Flow and Multi-Chamber Microfluidic Devices for Incubating Human Tissues
Author List:
Rabin, Allison; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Almada, Amanda; Undergraduate, Mechanical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Leclere, Michelle; Undergraduate, Mechanical Engineering, California Polytechnic State University, San Luis Obispo
Torres, Leah; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Heylman, Christopher; Faculty, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Abstract: Microfluidic devices are created to grow micron-scale human tissue in dynamic, 3D environments consisting of fluid channels and tissue chambers. This method mimics physiological conditions, providing a cost-efficient platform for drug screening and studying tissue microenvironments. “Tissues-on-a-chip” can potentially detect harmful side effects of drugs before their failure in preclinical or clinical trials. With funding from the CSUPERB President’s Commission Scholars Program, two microfluidic devices were created: a variable-flow device that simulates both normal and abnormal interstitial flow and a multi-chamber device that allows simultaneous trials to run through a single inlet and outlet, minimizing uncontrolled variables between tissues and maximizing efficiency. Device designs were created using computer-aided design software, SolidWorks, and steady-state flow was simulated in COMSOL Multiphysics software. For the variable-flow device, simulations showed manipulating flow rate at the inlet directly varied longitudinal fluid velocity through the tissue chamber. In the multi-chamber simulations, longitudinal fluid velocities through each chamber differed from each other by 0.2μm/s, and the flow between chambers was negligible (<0.01μm/s); this supports that fluid velocity will be similar between chambers and cell waste will flow through the device without contaminating adjacent chambers. Soft photolithography was used to imprint the designs onto biocompatible-polymer polydimethylsiloxane (PDMS), which was hole punched, then plasma bonded to glass slides, to create the devices. Simulation results were verified by pumping fluorescent microbeads through the multi-chamber device with a syringe pump. Video microscopy and ImageJ were used to characterize the velocities of five beads through the center of a chamber. The average velocity fell within 20.1% of the simulated value, supporting the feasibility of our simulated results. In the variable-flow device, a velocity range of 0.69-2.66μm/s was achieved empirically, which encompasses healthy interstitial range (0.75-1.25μm/s). Extracellular matrix (ECM) loading was tested using fluorescent fibrin gel. It was qualitatively confirmed that ECM remained within the chambers of both devices. This work demonstrates a proof of concept that proper flow and ECM loading can be achieved with these designs, enabling their use in 3D tissue culture for high throughput screening.
Poster #: 68
Campus: San Diego State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Microbiome, Flow cytometry, Indoor environment
Project Title: Quantitative and FACS analysis of bacterial and fungal communities in indoor environment.
Author List:
Tandon , Ruby ; Graduate, Biology, San Diego State University, Presenting Author
Smurthwaite , Cameron ; Graduate, Biology , San Diego State University
Abstract: Microorganisms present in indoor settings, where we spend ~90% of our time, impact human health and well-being. Deeper understanding of the built environment microbiome is needed to be able to efficiently manage these microorganisms. While, the overall diversity of indoor systems has been increasingly well characterized, the vast majority of the studies are not quantitative and do not distinguish between what microorganisms are alive and growing (the most important fraction) and those that are dead. Here, we used a multi-technique approach to build upon existing knowledge of the built environment and allow broader characterization of microbial taxa in fiberboard and drywall material in high moisture conditions. Using epifluorescence and differential contrast microscopy we estimated viral, bacterial and fungal abundance in the two material types. Fungi were almost exclusively observed on drywall coupons. We identified fungal genera: Alternaria, Ulocladium and Chaetomium on drywall coupons at later timepoints. These are known secondary and tertiary colonizers of building materials with high water activity. Fungal spores and hyphae length were estimated separately for the two material types. We observed a decline in the number of spores at later timepoints and simultaneous appearance of hyphae. This was most likely due to spore germination due to increased nutrient availability in the presence of water. These results demonstrated that drywall is more susceptible to water damage and microbial succession. Furthermore, we extended our analysis of the dynamics of microbial growth and metabolism on building materials by using flow cytometry-based methods to differentiate between viable and non-viable cells. The viable:non-viable ratios were found to be different for the two material types suggesting differential turnover of microbial cells as various species compete for space and resources. We also utilized Fluorescence Assisted Cell Sorting (FACS) to collect viable and non-viable cells separately and identified constituents using 16S rRNA sequencing. Combined with ITS sequencing to determine identity of fungal species, we will be able to study the nature of interaction in microbial communities. Overall, this study has important implications for buildings with leaks, in flood-prone regions, spaces with prolonged damp conditions like bathrooms and their impact on human health.
Poster #: 69
Campus: Sonoma State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: diabetic retinopathy, machine learning, embedded systems
Project Title: Low-Cost Diabetic Retinopathy Detection Solution using Machine Learning in Embedded Systems
Author List:
Manimala, Cheeku; Graduate, Engineering Science, Sonoma State University, Presenting Author
Faria, Cristin; Undergraduate, Engineering Science, Sonoma State University
Espinosa, Diego; Undergraduate, Engineering Science, Sonoma State University
Lawrence, Logan; Undergraduate, Engineering Science, Sonoma State University, Presenting Author
Shrestha, Sudhir; Faculty, Engineering Science, Sonoma State University
Abstract: Diabetic retinopathy is one of the leading causes of vision loss in the United States and around the world. People who have diabetic retinopathy may not have any symptoms until the condition becomes sever. Thus, the medically underserved populations are at increased risk of diabetes retinopathy related blindness. This poster will present our work towards developing a low-cost eye screening solution to help increase access to a regular eye exam to the medically unresolved populations. The system can be operated by a health technician with limited training. In the presented work, color fundus retina images obtained from publicly available diabetic retinopathy image databases were used for training and testing of machine learning algorithms. Out of 21 different classifiers tested, linear support vector machine (SVM) gave the best overall accuracy. Consequently, a set of 30 healthy and 30 diabetic retinopathy images were used for training and testing of the SVM. Testing was performed with a leave one out validation method where 59 images were used for training and the remaining image was used for testing. This was repeated until all images were used for testing. The trained algorithm was able to detect diabetic retinopathy images with 91.7% accuracy. Although the dataset was relatively small and further study is needed, it shows that diabetic retinopathy can be detected with a high accuracy using a trained SVM. Two NVDIA graphics processing unit (GPU)-based embedded systems known for their efficiency with machine learning and deep learning algorithms and a host machine necessary to train and test the embedded systems have been acquired. A low-cost digital ophthalmoscope has been identified for integration with the embedded systems. Ongoing efforts in this work includes implementing the trained algorithm on an embedded system environment and real-time testing with the images received directly from the retina imaging camera. The results, methods used, and the conclusions from the data will be presented and discussed.
Poster #: 70
Campus: Stanislaus State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Corrole, Spectroscopy, Solvent Effect
Project Title: Effect of Electron-Donating and -Withdrawing Groups in the Photophysical, Electrochemical, and Spectroelectrochemical Properties of Corrole Tautomers
Author List:
Webb, Kevin; Undergraduate, Biology, California State University, Stanislaus, Presenting Author
León, Aliz; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Raker, Robyn; Undergraduate, Chemistry, California State University, Stanislaus
Mendez, Suzanne; Undergraduate, Chemistry, California State University, Stanislaus
Alemán, Elvin; Faculty, 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. In addition, a proper understanding of corroles interaction with electron-donating and electron-withdrawing groups 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.
We investigated the photophysical, electrochemical, and spectroelectrochemical properties of substituted-free-base triphenyl corrole (Xn-H3TPCor). The long-term goal is to fully understand how electron-withdrawing (EW) and electron-donating (ED) groups attached to Xn-H3TPCor control the tautomerization and the photophysical, electrochemical, and spectroelectrochemical properties of corrole molecules. We characterized corroles ability to absorb and emit light in various solvents and in a multitude of concentrations, and the role of solvent in the fluorescence quantum yield. UV-Visible data shows that when strong EW groups are used, the photophysical properties are similar to those obtained for the unsubstituted oxidized species. However, no significant effects were observed when the ED group was used. The electrochemical and spectroelectrochemical data shows more complex profiles, compared to those obtained for unsubstituted corrole molecules. Many of the same properties that we found for unsubstituted free-base corrole and the substituted corroles have similar trends in regards to the solvent effect in the tautomerization of corrole. These results will help us to better understand how functional groups can affect the application of corroles in artificial light harvesting complexes.
Poster #: 71
Campus: CSU Los Angeles
Poster Category: Diagnostics/Imaging/Analytical
Keywords: resistive pulse, surface plasmon resonance microscopy, liposome
Project Title: Delivery of Single Liposomes from Micropipettes under Resistive-Pulse Control
Author List:
Chang, Megan; Undergraduate, Chemistry and Biochemistry , California State University, Los Angeles, Presenting Author
Morgan, Georgia; Undergraduate, Chemistry and Biochemistry , California State University, Los Angeles, Presenting Author
Wang, Yixian ; Faculty, Chemistry and Biochemistry , California State University, Los Angeles
Abstract: A resistive pulse based delivery system has been developed to precisely deliver single liposomes. In resistive pulse sensing, a potential is applied to drive ion flow through a pipette opening and the ionic current is monitored. When a charged particle is driven to pass through the orifice, partial blockage of the pipette orifice results in a current resistive pulse, which can be used to quantify and identify the size and surface charge of a single particle. In this work, liposomes with diameter ranging from 100nm to 500 nm were successfully delivered with 1.5 micrometer diameter micropipettes at a controlled frequency by optimizing the concentration of liposomes and the buffer condition. Individual resistive pulse were fit to simulation to extract the size and surface charge information for the corresponding liposome. Precise delivery of single liposomes to desired location was demonstrated by surface plasmon resonance microscopy (SPRM), which is extremely sensitive to surface events like delivered liposome binding to the substrate. This successful delivery model will be applied to study the localized structural disturbances caused by antimicrobial lipids coupled with high-resolution imaging techniques such as atomic force microscopy (AFM) and SPRM. We are also working on delivery of smaller particles such as single proteins and their aggregates for other biomedical applications. This research was supported by the CSULA Startup Fund, CSUPERB New Investigator Grant, and the National Institute of General Medical Sciences of the National Institute of Health under Award Number R25GM061331.
Poster #: 72
Campus: Stanislaus State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: kombucha, gas chromatography, ethanol
Project Title: Quantitation of ethanol in kombucha beverages by gas chromatography
Author List:
Hillberg, Lynsey; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Hernandez, Ruben; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Stessman, Nhu Y; Faculty, Chemistry, California State University, Stanislaus
Russell, Scott; Faculty, Chemistry, California State University, Stanislaus
Abstract: The purpose of this study was to determine the percent of alcohol by volume (ABV) in several commercial kombucha tea beverages and to determine whether or not bottle fermentation can lead to significant increases in ABV values. Kombucha is a popular tea to drink. It is reported to contain trace amounts of alcohol because it is produced by fermenting sweet tea with yeast and bacteria cultures. Kombucha tea is often marketed as a health drink rather than an alcoholic beverage because of its low volume percent of alcohol. However, the Alcohol and Tobacco Tax and Trade Bureau (TTB) website states, “Under federal law, if the alcohol content of kombucha is 0.5% or more alcohol by volume, at any time during production, when bottled, or at any time after bottling, the kombucha is an alcohol beverage and is subject to TTB regulations.”(1) In our study, gas chromatography was used to quantify the ABV of several commercially available kombucha teas. Standard solutions were prepared to generate standard curves correlating ethanol ABVs to chromatographic peak areas. 1-propanol was used as an internal standard in all samples. Kombucha samples represented two that were fruit-infused and one that was not. The fruit-infused kombucha teas had initial average ethanol ABVs of 1.79 (+/- 0.01)% and 1.4 (+/- 0.2)%. In contrast, to the pure tea version of a Kombucha beverage which was initially 0.57 (+/- 0.02) % ABV. These samples were then allowed to bottle ferment at room temperature and analyzed weekly. When left at room temperature for ~2.5 weeks the ABV of the fruit-infused kombucha teas increased to 2.8 (+/- 0.2)%, and 3.20(+/- 0.06)%, while the pure tea version only increased to 1.0 (+/- 0.1)%. This is not surprising since the fruit-infused kombucha teas likely had more residual fermentable sugar available for bottle fermentation. These findings underscore the need to clearly label these products and inform consumers to store them under refrigeration. These results also agree with another recently published gas chromatographic study on kombucha tea ABVs.(2) Future efforts will expand the analysis to a larger sample set in order to establish whether or not these findings are representative generally, or unique to the initial samples analyzed.
References
1. Alcohol Tobacco Tax and Trade Bureau Kombucha Page. https://www.ttb.gov/kombucha/index.shtml (accessed Sept. 17, 2018)
2. Ebersole, B.; Liu, Y.; Schmidt, R.; Eckert, M.; Brown, P. J. AOAC Int. 2016, 100, 732-736
Poster #: 73
Campus: CSU Los Angeles
Poster Category: Diagnostics/Imaging/Analytical
Keywords: microfluidics, electrochemical detection, glucose sensor
Project Title: Microfluidic Thread-Based Electrode System to Detect Glucose and Acetylthiocholine
Author List:
Uchida, Kathryn; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Gaines, Michelle; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles
Gonzalez-Guerrero, Maria Jose; Postdoc, Chemistry and Biochemistry, California State University, Los Angeles
Gomez, Frank A.; Faculty, Chemistry and Biochemistry, California State University, Los Angeles
Abstract: The rapid growth in technology has greatly enhanced the scope of real-time health monitoring and has facilitated the shift of healthcare services from clinic-centric to patient-centric. Portable devices and systems that enable medical diagnostics outside of traditional laboratory settings are increasingly a part of a patient’s home healthcare regimen. Herein is described a reusable and simple to fabricate electrochemical sensor for the detection of glucose and acetylthiocholine using thread-based electrodes and nylon thread. The fabrication of the device consisted of two steps. First, three nylon-based electrodes (reference, working, and counter) were painted with one layer of conductive inks (silver and carbon ink, or silver/silver chloride ink). The electrodes were taped onto parafilm, and a piece of white nylon thread was wrapped around each electrode connecting the three electrodes. For the glucose system, a phosphate buffered saline (PBS) solution containing glucose oxidase (GOx) (10 mg/mL), and potassium ferricyanide (K3[Fe(CN)6]) (10 mg/mL) as mediator, was dried onto the thread, and increasing concentrations of glucose (0-15 mM) was added to the thread and measured by cyclic voltammetry (CV). The current output from the glucose oxidation was proportional to the concentration of glucose. For the second system, a solution of acetylcholinesterase (AChE) (0.08 U/mL) in PBS was added to the nylon thread, and increasing concentrations of acetylthiocholine (ATC) (0-9.84 mg/mL) was added and measured by CV. The current output from the oxidation of thiocholine (produced by AChE reacting with ATC) was proportional to the concentrations of ATC added to the thread. From both systems, a graph of current output vs. substrate concentration was produced and fitted with a linear regression line that gave R2 values of 0.985 (GOX/glucose) and 0.995 (AChE/ATC). Production costs of the sensors are lower than other devices since the materials are inexpensive, reagent requirements are minimal, and the sensors are reusable. These advantages make them well suited for resource-limited regions throughout the world where access to state-of-the-art POC diagnostic devices is not always available. Future work is focused on modifying the design to improve the detection capabilities of other analytes, thereby, broadening the use of thread-based applications with the goal of having a positive impact on the health and well-being of society.
Poster #: 74
Campus: CSU Los Angeles
Poster Category: Diagnostics/Imaging/Analytical
Keywords: microfluidics, ELISA, assays
Project Title: Cord-Based Microfluidic Chips as a Platform for ELISA and Glucose Assays
Author List:
Elomaa, Jenny; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Gallegos, Laura; Graduate, Chemistry and Biochemistry, California State University, Los Angeles
Gomez, Frank A.; Faculty, Chemistry and Biochemistry, California State University, Los Angeles
Abstract: Since the development of the first microfluidic devices (MDs) in the early 1990s, microfluidic technologies have evolved to incorporate a wide range of applications. The most promising real-world application has been point-of-care (POC) diagnostics. POC testing has the potential to greatly improve health care and in resource-limited settings where access to quality medical diagnosis and proper medication may not always be available. The past decade has seen the development of various substrates to supplant traditional glass and polymeric-based platforms including paper and thread that can serve as inexpensive substrates for POC and biosensor applications. Herein, we describe the development and application of microfluidic cord-based analytical devices (µCADs) in two enzyme-linked immunosorbent assays (ELISAs) and glucose assay. Here, the quantitative detection of biotinylated goat anti-mouse immunoglobulin (IgG) antibody, rabbit IgG antibody, and glucose via colorimetric analysis is detailed. In the ELISA systems, antibody is spotted on the detection site and subjected to a series of washes, addition of streptavidin-alkaline phosphatase (Strep-ALP) or alkaline phosphatase (ALP)-conjugated secondary antibody, and colorimetric substrate. The devices are scanned and analyzed yielding a correlation between inverse yellow or inverse blue intensity, and antibody concentration. For the first ELISA, a linear range of detection is observed at lower concentrations (2.50 X 10-4–1.75 X 10-3 mg/mL) of Strep-ALP with saturation of the enzyme achieved at higher concentrations (>2.50 X 10-4 mg/mL). For the second ELISA, the L50 was demonstrated to be 167.6 fmol/zone. The glucose assay consisted of spotting increasing concentrations of glucose on the analysis sites and transporting, via capillary action, a solution mixture of glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI) to the sites whereby a yellow-brown color is observed indicating oxidation of iodide to iodine. The device was then dried, scanned, and analyzed yielding a correlation between yellow inverse intensity and glucose concentrations. Studies to quantitate glucose in artificial urine showed good correlation with the µCAD. Future work will focus on fabricating new diagnostic devices that can be used in the growing healthcare industry in resource-limited settings and developing countries.
Poster #: 75
Campus: CSU Sacramento
Poster Category: Disease (Pathogens)
Keywords: Fluorescence In Situ Hybridization, Microbiome, Inflammation
Project Title: Characterizing Shifts in Microbiome Biogeography Mediated by Inflammatory Skin Disorders
Author List:
Truong, Catalina; Graduate, Biological Sciences, California State University, Sacramento, Presenting Author
Peterson, Stephanie; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Kumar, Sheelta; Graduate, Biological Sciences, California State University, Sacramento
Haas, Kelly; Postdoc, Biological Sciences, California State University, Sacramento
Sivamani, Raja; University of California Davis
Crawford, Robert; Faculty, Biological Sciences, California State University, Sacramento
Abstract: Interest in studying microbial diversity and function in health and dysbiosis has emerged thanks in part to technologies facilitating elucidation of phylogenetic signatures of the human microbiome. One clinical characterization remaining largely unknown is the composition of microbes in sebaceous glands associated with inflammatory skin disorders such as acne and rosacea, which are commonly treated by topical administration of antibiotics and steroids. Here we use next generation high throughput metagenomic sequencing (NGS) and fluorescence in-situ hybridization (FISH) to identify species diversity and functional gene clusters in follicular casts obtained from human subjects at the UC Davis Medical Center. Briefly, affected and control tissues were obtained from patients with acne or rosacea using strips that extract 6 – 8 deep skin follicles per sampling event. The total microbial genomic DNA was extracted from follicular pools for each grouping of inflamed tissue and analyzed for relative phylogenetic abundance. Our results show an overrepresentation of Cutibacterium acnes, Corynebacterium granulosum, Staphylococcus aureus, Propionibacterium humerusii, and Propionibacterium avidum at inflamed versus control sampling sites, and that the relative abundance of each species changed with differential lysozyme incubation and heat shock steps during DNA extraction. Additionally, we developed DNA probes to visualize microbiome biogeography by confocal scanning laser microscopy within fixed follicle tissues using FISH analysis. Our results confirm the microbial diversity observed by genomic sequencing methodologies and present one of the first clinical depictions of shifting microbiota in deep follicle tissues affected by inflammatory skin disorders. A better understanding of microbial biogeography using long-term longitudinal studies and optimized methods for extracting DNA and visualizing microbes promises to elucidate novel non-antibiotic and non-steroid treatment strategies for combatting microbiome shifts in acne, rosacea, and related dysbioses. These studies represent collaborative efforts between UC Davis and CSUS and were supported by generous gift funding from Banyan Botanicals.
Poster #: 76
Campus: CSU Fullerton
Poster Category: Disease (Pathogens)
Keywords: Trypanosoma cruzi, Ion channels, CRISPR-Cas9
Project Title: Evaluation of structure-function of a mechanosensitive channel in Trypanosoma cruzi.
Author List:
Tiwari, Megna; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Foster, Kyle; Undergraduate, Biological Science, California State University, Fullerton
Dave, Noopur; Graduate, Biological Science, California State University, Fullerton
Jimenez, Veronica; Faculty, Biological Science, California State University, Fullerton
Abstract: In order to complete its life cycle, Trypanosoma cruzi-the protozoan parasite that causes Chagas disease- must adapt to drastic environmental changes as it moves from an insect vector to a mammalian host. We have identified and characterized a bacterial-like mechanosensitive channel in T. cruzi (TcMscS). TcMscS, has shown to participate in sensing environmental changes in the parasite by mediating the flow of ions and osmolytes across membranes. Previous studies in our lab have shown that CRISPR/Cas generated knock out (KO) epimastigote cell lines have a defective growth rate, lower infectivity, and decreased intracellular replication when compared to wild type (WT) epimastigotes. Computational 3D simulations of TcMscS structure have allowed us to identify key residues that could be playing a role in regulating the activity of the channel. To test this hypothesis, we have obtained gain of function and loss of function mutants by site directed mutagenesis. The mutants have been cloned in pTREX vector and transfected into WT and TcMscS-KO epimastigotes to examine the phenotype of the complemented strains. We have evaluated osmoregulation, replication, infectivity and growth. Additionally, we have complemented the TcMscS-KO cell line with the ortholog of the channel from Trypanosoma brucei (TbMscS) to examine if there is a reversion of phenotype. Mechanosensitive channels such as TcMscS are not found in animals and share low homology with mammalian proteins, making them a suitable target for development of new therapeutics that could minimize detrimental side effects observed with the currently available drugs to treat patients with Chagas disease.
Funding: AHA 16GRNT30280014 and NIH R15 AI122153.
Poster #: 77
Campus: CSU Sacramento
Poster Category: Disease (Pathogens)
Keywords: Glycodendrimers, HIV, MST
Project Title: Synthesis, Optimization and Analysis of Hexavalent Sulfoglycodendrimers as Anti-Viral Agents
Author List:
Vierra, Cory; Graduate, Department of Chemistry, California State University, Sacramento, Presenting Author, Eden Award Nominee
McReynolds, Katherine; Faculty, Department of Chemistry, California State University, Sacramento
Abstract: Research in the McReynolds lab focuses on the synthesis of dendrimers. These molecules consist of an organic core of varying branch number, and a variety of functional groups available on the surface. The ability of dendrimers to exhibit the multivalent effect makes them prime candidates for study. Multivalency refers the simultaneous attachment of multiple binding sites on one entity with multiple receptors on another. When studied, glycosylated dendrimers, or glycodendrimers, have shown inhibitory activity towards viral binding. This study focuses on the syntheses of sulfoglycodendrimers (SGD’s) and the evaluation of their anti-viral properties.
In present research, a hexavalent dendrimer core was synthesized through a four-step divergent pathway. The core was synthesized through the branching of tris(2-aminoethyl)amine, a trivalent core, using methyl acrylate to form a hexavalent dendrimer, followed by the addition of 1,3 diaminopropane to extend the dendrimer arms, resulting in 83% and 95% yields, respectively. Next, the core was subjected to an amide coupling of Boc-(aminooxy)acetic acid, and finally deprotection, resulting in a 54% (two step) yield. The dendrimer core was next glycosylated in a laboratory grade microwave under acidic conditions using discrete length oligomers of colominic acid ranging from monomer through tetramer to generate four novel glycodendrimers. Once confirmed via standardized gel permeation chromatography (GPC), the glycodendrimers were sulfated to give SGD’s
The SGD’s will be next analyzed in three unique ways to determine their efficacy as antiviral agents. To begin, an initial in house in-vitro enzyme-linked immunosorbent assay will be performed to analyze binding between gp120 and each SGD. After binding confirmation, an in-vitro live-cell HIV-1 neutralization assay will be conducted to analyze the antiviral characteristics as well as any cytotoxicity through a collaboration with Dr. Celia LaBranche at Duke University. Finally, microscale thermophoresis (MST) will be conducted at San Jose State, with the help of Dr. Daryl Eggers, to determine binding strength (KD) between the viral protein of interest, gp120, and each SGD. The antiviral capability of the compounds generated in this study will be directly compared to other ongoing projects within the lab to guide further SGD development. It is believed that the SGD’s generated here will have significant inhibitory antiviral characteristics.
Poster #: 78
Campus: CSU Chico
Poster Category: Disease (Pathogens)
Keywords: influenza virus, macrophage,
Project Title: H5N1 Influenza Viruses Alter Macrophage Function in a Replication-Dependent Manner
Author List:
Beck, Donald; Graduate, Biological Sciences, California State University, Chico, Presenting Author
Soto, Armando; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author
Cline, Troy; Faculty, Biological Sciences, California State University, Chico
Abstract: H5N1 influenza viruses cause human infections with a high mortality rate. However, the mechanism of enhanced disease severity is incompletely understood. Macrophages play a critical role in protection against influenza viruses yet have been implicated in enhanced disease severity following H5N1 infection. H5N1 influenza viruses are unique in their ability to replicate in macrophages, a feature that maps to the viral hemagglutinin (HA) gene. We hypothesize that H5N1 influenza viruses cause severe disease by altering macrophage functions. Macrophages have been classified as classically-activated (M1) or alternatively-activated (M2) as defined by the cytokines that drive macrophage activation and the gene expression profile of the polarized macrophage. M1 macrophages are pro-inflammatory, while M2 macrophages are involved in tissue repair. We investigated the impact of influenza virus infection on macrophage polarization by infecting macrophages with the H1N1 virus CA/09 (unable to replicate in macrophages) or with CA/09 expressing an H5 HA gene (CA/09+H5 HA; replicates in macrophages). Cells infected with CA/09 expressed both iNOS (inducible nitric oxide synthase) and MMR (macrophage mannose receptor; markers of an M1 and M2 polarization, respectively), suggestive of a balanced M1/M2 response important for recovery from infection. Infection with CA/09+H5 HA induced expression of MMR only, indicating a polarization toward the M2 phenotype. Further, macrophages infected with the CA/09+H5 HA were not responsive to an M1-polarizing stimulus (IFN-γ) while remaining responsive to the M2 stimulus, IL-4. These results suggest that influenza viruses modify macrophage functions in a strain-dependent manner correlating with replication of the virus in macrophages.
Poster #: 79
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: Pam3CAG, innate immunity, influenza
Project Title: Cross-Protective Response Against H1N1 and H3N2 Influenza Challenge Following Vaccination of Mice with Liposomes Containing only the Adjuvant Pam3CAG
Author List:
Faneuff, Eden; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Kunihiro, Connor; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Adler-Moore, Jill; Faculty, Biological Sciences, California State Polytechnic University, Pomona
Abstract: Introduction: Current influenza vaccines depend upon stimulation of the adaptive immune response to viral proteins that mutate year to year, making it difficult to predict with certainty which viral proteins need to be incorporated into the yearly influenza vaccine. Another approach to address this problem could be to stimulate an innate immune response that would be effective against different viral strains. In this study, we used a liposomal carrier to deliver a potent innate immune stimulating adjuvant, Pam3CAG, to mice, prior to challenge with different influenza strains.
Methods:We used a liposomal vaccine made by Molecular Express Inc.(CALV VesiVax liposomes)that included the lipophilic Pam3CAG in the liposome bilayer(L-Pam3CAG). Swiss Webster mice were sedated intraperitoneally with 80mg/kg ketamine and 16mg/kg xylazine and intranasally(IN) given one of the following L-Pam3CAG regimens (25ug Pam3CAG/dose) prior to viral challenge on d0: d-4/d-2 vaccinations, d-2 vaccination, d-4 vaccination, d-4 buffer control. On d0 mice were challenged IN with 10XLD50 H1N1 (PR/8 strain) or H3N2 (X-31 strain) and 8 mice/group were monitored for morbidity to d+20. On d+5, lungs from other H1N1 challenged mice were collected (5 mice/group) to assess viral burden via a foci infection assay.
Results: Survival was significantly better for mice given L-Pam3CAG on d-4 for H1N1 (75%, p=0.04) and H3N2 (100%, p=0.0038) versus PBS mice (25% and 28% survival, respectively). Weight change paralleled survival, with H1N1 or H3N2 challenged mice losing significantly less weight than their respective PBS groups (for H1N1 d-4, p=0.0047 and d-4/-2 p=0.0289 and for H3N2 d-4, p=0.0019 and d-2, p=0.049). Disease signs also paralleled survival with H1N1 or H3N2 challenged mice having significantly lower disease signs than their respective PBS groups (for H1N1 d-4/-2, p=0.0006 and for H3N2 d-4, p=0.0087 and d-2, p=0.0054). There was significantly less viral burden in the lungs of H1N1 mice given L-Pam3CAG on d-4 versus mice given PBS (p=0.0317) or mice given L-Pam3CAG d-4/-2 (p=0.0079).
Conclusions: The ability of the L-Pam3CAG to stimulate a protective innate immune response against both H1N1 and H3N2 influenza strains, when the vaccine was given only on d-4 relative to challenge, suggests that this short treatment has the potential to provide needed protection in case the viral proteins of a given circulating influenza strain have not yet been determined.
Poster #: 80
Campus: San Diego State University
Poster Category: Disease (Pathogens)
Keywords: cancer immunotherapy, mouse model of melanoma, metastatic prevention
Project Title: A novel anti-cancer immunotherapy, VAX014, is oncolytic and induces tumor regression in a mouse model of melanoma
Author List:
Reil, Katherine; Graduate, Biology, San Diego State University, Presenting Author
Tsuji, Shingo; Vaxiion Therapeutics Inc.
Giacalone, Matthew; Vaxiion Therapeutics Inc.
McGuire, Kathleen; Faculty, Biology, San Diego State University
Abstract: Immunotherapeutic agents that are both immune-stimulating and toxic to tumor cells prevent tumor growth, recurrence, and metastasis. Aggressive cancer types such as melanoma have a high propensity to metastasize and localized treatments that halt disease progression are desperately needed. Vaxiion Therapeutics has developed a novel E. coli derivative called recombinant bacterial minicells (rBMCs) that target and deliver therapeutic molecules directly to cancer cells. VAX014 rBMCs (VAX014) have been engineered to be a tumor-targeted oncolytic agent by incorporating invasin, an rBMC surface-localized protein that targets α3β1 and α5β1 integrins expressed on tumor cells, and perfringolysin O (PFO), a bacterial toxin that facilitates tumor cell membrane permeabilization and cell death.
To determine if VAX014 is a potential therapy for localized intralesional treatment of melanoma, its activity is being tested in the syngeneic B16F10 melanoma model. First, in vitro experiments using flow cytometry (FC) confirmed that B16F10 express cell surface α3, α5, and β1 integrins, while a standard cell-killing potency assay demonstrated sensitivity of B16F10 cells to VAX014. A combination of lactate dehydrogenase release and propidium iodide uptake assays then confirmed PFO-dependent membrane permeability. VAX014-treated B16F10 cells also demonstrated hallmarks of apoptosis including pyknotic nuclei and a loss of mitochondrial membrane potential. These data all support that VAX014 mediates cell membrane permeabilization (oncolysis) and concomitant apoptosis in B16F10 cells.
Since VAX014 exhibits oncolytic activity and cytotoxicity against B16F10 in vitro, a series of in vivo anti-tumor efficacy experiments have been initiated on intradermal B16F10 lesions in mice. In early dose range studies, intralesional VAX014 injections induced either partial or full tumor regression (p<0.005) and enhanced survival (p<0.05). Follow-on dose frequency experiments have determined the most effective dosing (1x/week) and concentration levels (1.5x108 VAX014/dose). In ongoing experiments, tumor-infiltrating immune cell populations and immunophenotypes will be identified using FC and immunohistochemistry. Additionally, surviving animals will be re-challenged with B16F10 and left un-treated to test for systemic anti-tumor immunity. To date, B16F10 is responsive to VAX014 in vivo and further studies are being conducted to support clinical investigation of VAX014 in melanoma patients.
Poster #: 81
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: HSV-2, Pam3CAG, liposomes
Project Title: Evaluation of the Pam3CAG adjuvant in Herpes Simplex Virus Type 2 gD3pep Liposomal Vaccine
Author List:
Gonzalez, Edgar; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Adler-Moore, Jill; Faculty, Biological Sciences, California State Polytechnic University, Pomona
Abstract: Herpes simplex virus type 2 (HSV-2) causes sexually transmitted genital infections that are lifelong and result in recurrent outbreaks due to establishment of latency in neurons. More than 500 million people worldwide are infected with HSV-2, and HSV-2 infections increase the risk of contracting HIV-1. While anti-viral drugs reduce the frequency and severity of symptoms, no vaccine exists to prevent infections. Previous vaccine studies using conjugatable adjuvant lipid vesicles (CALV) with gD3pep, a peptide composed of three immunogenic segments of the HSV-2 surface glycoprotein D, have shown effective protection against HSV-2 infection when the vaccine has the MPL adjuvant, a TLR4 ligand. In this study, we investigated a CALV liposome formulation with gD3pep covalently conjugated with maleimide to the liposomes, containing the lipophilic adjuvant Pam3CAG, a synthetic analogue of E. coli lipoprotein that stimulates toll-like receptor 2. Phosphate buffered saline (PBS), gD3pep alone, gD3pep conjugated to CALV liposomes with no adjuvant, and unconjugated gD3pep mixed with CALV liposomes without adjuvant were tested as controls. Mice (n=17/group) received vaccine subcutaneously d0, d14, and d26. Serum and spleens were harvested d31 (n=5/group) for anti-gD3pep IgG serum isotype determination, and splenocyte cytokine secretion. Mice (n=12/group) were challenged intravaginally d35 with HSV-2 and monitored for morbidity for 28 days. Viral burden was determined on vaginal swab samples collected d37 (n=7/group) and spinal cord samples collected d41 (n=5/group) using a plaque forming unit (PFU) assay. The Pam3CAG and conjugated gD3pep had 29% survival versus unconjugated gD3pep and PBS with 0% survival (p< 0.01). Disease signs and weight loss paralleled survival. HSV-2 virus was undetectable in spinal cords and vaginal swabs of the Pam3CAG group versus viral infection in the vaginal swabs and spinal cords of conjugated gD3pep, unconjugated gD3pep and PBS control groups (200-400 PFU/ml vaginal swab and 1880-5500 PFU/g spinal cord). Immunologically, a predominantly T-helper 1 (Th1) response was stimulated by the Pam3CAG group based on antibody and cytokine assays. Overall, this data supports conjugating the gD3pep to the liposomes, with improved efficacy when the adjuvant Pam3CAG is associated with the liposomes.
Poster #: 82
Campus: CSU Fullerton
Poster Category: Disease (Pathogens)
Keywords: hemocyte, Drosophila, reactive oxygen species
Project Title: Rab8, a small GTPase, plays a regulatory role in Drosophila hemocyte activation during immune response.
Author List:
Harris, Caitlin; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Roshandell, Mobina; Graduate, Biological Science, California State University, Fullerton
Brennan, Catherine; Faculty, Biological Science, California State University, Fullerton
Abstract: During an immune invasion, phagocytic white blood cells are quickly activated to engulf microbes and form a phagosome. The phagosome then matures and becomes a toxic environment, effectively killing the microbes. This activation is characterized by an increase in free radical production and bacterial killing, as well as an upregulation of signaling pathways. Free radicals, such as reactive oxygen species (ROS), are known to kill microbes directly but may also be involved in signaling during an immune response. These ancient mechanisms are evolutionarily conserved between Drosophila hemocytes and mammalian macrophages and are well studied. However, how phagocyte activation is regulated is still unclear. Rab proteins are small GTPases that regulate cell signaling through membrane trafficking and recycling. Although several Rab proteins are known to regulate phagosome maturation during immune response, Rab8’s involvement in hemocyte activation and signaling has yet to be characterized. Utilizing dihydroethidium (DHE), a fluorescent ROS indicator, we found that rab8 mutants have an increased production of ROS during bacterial infection when compared to wild-type flies. Additionally, using plate count assays, we see that rab8 mutants have an accelerated rate of bacterial killing when compared to wild-type flies. This indicates that Rab8 may have a regulatory role in hemocyte activation. Since rab8 mutants display increased hemocyte activation, the normal role of Rab8 may be to slow or dampen the immune response. Further experiments will be conducted to understand the cellular events that link Rab8 trafficking and hemocyte activation.
Poster #: 83
Campus: CSU Los Angeles
Poster Category: Disease (Pathogens)
Keywords: Acinetobacter baumannii, gene knock out, Galleria melonella
Project Title: The effects of tonB and entA unmarked knockouts on the virulence of Acinetobacter baumannii LAC-4 in a Galleria melonella bacterial infection model
Author List:
Viramontes, Yesica; Undergraduate, Department of Biological Sciences, California State University, Los Angeles, Presenting Author
Madrid, Athena; Faculty, Department of Biological Sciences, California State University, Los Angeles
Ewing, Peter; Faculty, Department of Biological Sciences, California State University, Los Angeles
Xu, Howard; Faculty, Department of Biological Sciences, California State University, Los Angeles
Abstract: Acinetobacter baumannii is a Gram-negative bacterium that is an emerging threat due to its multi-drug resistance (MDR). One clinical isolate obtained from a Los Angeles County Hospital, LAC-4, has been found to be hypervirulent to mice and possess gene clusters conferring heme and iron acquisition capabilities. Previous work in our laboratory has shown deleting potential virulence genes in LAC-4 can be used to elucidate the role of genes to the bacterium’s hypervirulence. Such experiments on mutant strains are typically carried out in a mouse model of bacterial pneumonia. In order to minimize the use of mice in pathogenesis studies, preliminary in vivo assays may be carried out in a Galleria melonella waxworm model of bacterial infection. In this study we demonstrate how a TonB receptor mutant’s virulence is reduced in the Galleria melonella model in comparison to wild-type LAC-4. Additionally, we present our findings on an entA mutant and its complimented entA neutral-site knock-in strain in Galleria melonella. Both tonB and entA genes are parts of the heme/iron utilization gene clusters. To accomplish this, unmarked mutants were generated by cloning flanking regions to the relavent genes and stitching them together with an apramycin cassette using overlap extension PCR. Similarly, flanking regions to a neutral site were created and stitched around gene of interest, entA, which was then linked to an apramycin resistance gene to create a knock-in cassette. Constructs were then ligated into the suicide vector pMo130, transformed into E. coli DH5α and recombinant plasmid subsequently electroporated into A. baumannii LAC-4. Successful co-integrants were passaged to enrich for mutants that had undergone the second-crossover event. Mutants were screened and confirmed via DNA sequencing. Mutant and compliment strains were introduced into Galleria melonella via injection of approximately 5 x 10^5 CFUs of the appropriate strain into the last right pro-leg. Survival of the waxworms was monitored, and survival curves were graphed using the Kaplan-Meier method with differences in survival rates determined via log-ranked test. Our results demonstrated that the tonB and entA mutants showed significant difference in the rate of infection. Additionally, the knock-in complement of the entA mutant showed restoration of the wild-type phenotype. These results will facilitate further studies on the roles of TonB and EntA in the virulence of A. baumannii.
Poster #: 84
Campus: CSU Long Beach
Poster Category: Disease (Pathogens)
Keywords: fecal coliforms, antibiotic resistance, beaches
Project Title: Comparison of multiple antibiotic resistance in coliforms at Southern California beaches with differing wave action during dry and wet weather
Author List:
Hernandez, Rebecca; Graduate, Biological Sciences, California State University, Long Beach, Presenting Author
Acedo, Ismael; Undergraduate, Biological Sciences, California State University, Long Beach
Dillon, Jesse; Faculty, Biological Sciences, California State University, Long Beach
Abstract: Antibiotic resistance is a emerging, critical issue in human health today. Fecal indicator bacteria such as coliforms are important markers of contamination in public waters and have been reported as antibiotic-resistant in some settings, but little is known about their importance at California beaches. Since many people frequent Southern California beaches, it is important to know if/when antibiotic-resistant bacteria are most prevalent. This project is examining the incidence of multiple antibiotic-resistant (MAR) coliforms in Southern California beaches with differing wave action during wet and dry weather. Samples were collected from beaches with restricted wave action (Cabrillo Beach inside the Port of LA breakwater, Mother’s Beach in Long Beach, and Mother’s Beach in Newport Beach) and from sites without restricted wave action (Cabrillo Beach outside the breakwater, Seal Beach, and Newport Beach) during wet and dry weather. To select for total coliforms, water samples were membrane filtered and plated on mEndo Agar. Total coliforms were enumerated; twenty randomly selected isolates at each location were subject to antibiotic resistance testing via Kirby Bauer and MAR index was calculated. Total coliforms were most elevated at sites with restricted wave action during wet weather. Overall, resistance to ampicillin was most common and the highest MAR index (indicating highest level of MAR) was observed at sites with restricted wave action during wet weather. Mother’s Beach in Newport Beach had the highest overall MAR index during wet weather, so isolates from this location were tested using the minimum inhibitory concentration (MIC) method to compare if the degree of resistance to ampicillin differs in wet/dry weather sample isolates. Data collected so far show that ampicillin MIC is higher among isolates collected during wet weather at Mother’s Beach in Newport Beach. This project highlights that rain results in increased levels of total coliforms and indicates that swimming in sites with restricted wave action during wet weather carries the highest risk of exposure to MAR coliforms, although widespread multi-drug resistance was not observed. We would like to thank the CSU Council on Ocean Affairs, Science, and Technology for their generous funding supporting this work.
Poster #: 85
Campus: Cal Poly San Luis Obispo
Poster Category: Disease (Pathogens)
Keywords: Escherichia coli, antibiotic resistance, ethinylestradiol
Project Title: Exposure to hormonal contraceptives increases efflux and antibiotic resistance in Escherichia coli strains
Author List:
Clatch, Allison; Graduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author, Eden Award Finalist
Van Otterloo, Leah; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
VanderKelen, Jennifer; Faculty, Biological Sciences, California Polytechnic State University, San Luis Obispo
Yep, Alejandra; Faculty, Biological Sciences, California Polytechnic State University, San Luis Obispo
Abstract: The World Health Organization classifies antibiotic resistance as one of the most important public health threats of this century. It has been shown that resistance arises through accumulation of mutations following exposure to sublethal concentrations of antibiotics. However, the effects of other drugs on antibiotic resistance are less understood. Hormonal contraceptives (HCs) are one of the most common forms of birth control and have been shown to have an inhibitory effect on Escherichia coli’s growth. In addition, steroid hormones (the basis of HCs) have been shown to be substrates for efflux pumps. We hypothesized that exposure to HCs induces increased activity of efflux pumps to remove the hormone from the cytoplasm. As efflux pumps are broad range and nonspecific transporters, increases in their activity may correlate with increased removal of antibiotics from the cell, leading to resistance. To test this hypothesis, six commensal or uropathogenic E. coli strains were exposed to the estrogen in HCs, ethinylestradiol (EE), and subsequently tested for their ability to accumulate the fluorescent dye Hoechst (H) 33342, a known substrate of efflux pumps. Compared to unexposed cells, E. coli incubated with EE accumulated significantly less H33342, suggesting an increase in efflux induced by the hormone. To investigate whether more efflux resulted in improved resistance to antibiotics, we tested the E. coli strains exposed to EE for growth in chloramphenicol, also a known substrate of efflux pumps. Cells pre-exposed to 100 μg/mL EE for 105 min were able to grow in the presence of chloramphenicol at concentrations close to the minimal inhibitory concentration, whereas no growth was observed for the unexposed cells. Finally, we tested whether these effects were due to increased activity of the best studied efflux pump, AcrAB-TolC. Quantitative PCR analysis as well as H33342 accumulation and growth in the presence of chloramphenicol all indicate that the AcrAB-TolC system is only partially responsible for the observed effects. Ongoing experiments are aimed at identifying other mechanisms by which EE induces increased efflux and antibiotic resistance. To the best of our knowledge, this is the first report of an increase in antibiotic resistance in gut bacteria following exposure to a component of hormonal birth control. Furthermore, our results suggest that common pharmaceuticals other than antibiotics can increase antibiotic resistance.
Poster #: 86
Campus: CSU Sacramento
Poster Category: Disease (Pathogens)
Keywords: Curcumin, Biofilm, Microbiota
Project Title: Curcumin-Mediated Modulation of Bacterial Communities in Inflammatory Skin Dysbiosis
Author List:
Tran, Jennifer; Graduate, Biological Sciences, California State University, Sacramento, Presenting Author
Valdez, Nico; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Crawford, Robert; Faculty, Biological Sciences, California State University, Sacramento
Abstract: Dysbiosis of the skin microbiome and underlying immune response to microbial antigens contributes to the pathogenesis of atopic dermatitis and several other disorders. DNA sequencing analysis has shown that bacterial communities of human patients shift to a concurrent overabundance of pathogenic Staphylococcus aureus (SA) and reduction of commensal Staphylococcus epidermidis (SE) during bouts of chronic inflammation relapse and a return to greater overall diversity following antibiotic treatment. However, antibiotic resistance and person-to-person variability in microbial communities is rendering currently prescribed therapeutic regimens unsuccessful, thereby highlighting a paucity in novel remediation approaches. To that end, food derivatives including curcumin have been explored for prebiotic intervention as a means of shifting bacterial populations based on antimicrobial and/or metabolic properties, but published studies present conflicting evidence toward efficacy and the underlying mechanisms remain unclear. Here we use a combination of physiological and molecular experiments to show that curcumin increases phenotypes associated with commensal SE but not pathogenic SA including growth rate and biofilm formation by increasing expression of genes previously shown to be important for adherence and extracellular matrix production. Additionally, curcumin dispersed existing biofilm formation for SA but not SE and transcriptomic analysis showed differential metabolism profiles in planktonic and biofilm-associated populations. Lastly, short chain fatty acids derived from SE cultures and biofilm communities generated less lipid and inflammatory cytokine production in an in vitro cell culture model system. Collectively, these results suggest the use of curcumin for remodeling microbial communities toward a predominance of non-pathogenic organisms. These studies represent collaborative efforts between UC Davis and CSUS and were supported by generous gift funding from Burt’s Bees.
Poster #: 87
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: Cannabinoid Receptors, Candida albicans, Sex
Project Title: The role of cannabinoid receptor (〖CB〗_2 R) and sex during an acute infection with Candida albicans in mice
Author List:
Janilkarn-Urena, Isis; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Buckley, Nancy; Faculty, Biological Sciences, California State Polytechnic University, Pomona
Abstract: Currently, 30 states and the District of Columbia have enacted laws legalizing marijuana for therapeutic and in some states, recreational purposes. The prevalence rates of marijuana use among US adults more than doubled since 2001. Unfortunately, the effects of marijuana on health, specifically on the immune system, are not well understood. Δ9 –Tetrahydrocannabinol (THC) is the principal psychoactive component of marijuana. THC shares the ability with endogenous cannabinoids, anandamide and 2-arachidonoylglycerol, to activate cannabinoid receptors. Central cannabinoid receptors (CB1R) are highly expressed in the central nervous system, while the peripheral cannabinoid receptors (CB2R) are mainly expressed in immune cells, implicating CB2R in immunomodulatory functions. Studies show that THC suppresses mouse resistance to certain bacterial, viral and protozoan infections. Our lab has reported THC having a suppressive effect on resistance to a secondary systemic infection by Candida albicans (C. albicans) in female mice. C. albicans is a fungus that resides in the human flora and can become pathogenic in immune compromised individuals with mortality rates up to 50% when infections become systemic. The roles of CB2R and sex on mouse resistance to fungal infections are unknown and have been the focus of the present work. Wild type (CB2R+/+) and CB2R KO (CB2R-/-) male and female mice were injected intravenously with one million C. albicans cells/mouse. Cytokines were analyzed from blood serum and splenocytes and tissue C. albicans load was measured 3 days after infection. Preliminary results show that tissue fungal loads were comparable between male and females regardless of genotypes. Preliminary results also show that infected CB2R+/+ male mice are more susceptible to the infection than CB2R-/- male and CB2R+/+ female mice as assessed by mortality and weight loss. Infected CB2R-/- male and female mice have comparable survival rates, although the females lost more weight than the males. Splenocytes from CB2R+/+ female mice challenged in vitro with heat killed C. albicans secreted less TNF-α compared to that from CB2R+/+ male mouse splenocytes. However, splenocytes from CB2R-/- female mice challenged in vitro with heat killed C. albicans secreted more TNF-α compared to that from CB2R+/+ male mouse splenocytes. These results suggest that CB2R and sex play a significant role in mouse response to systemic C. albicans infection.
Poster #: 88
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: blood, zebrafish, stem cells
Project Title: ccl44: A necessary gene for hematopoietic progenitor cells
Author List:
Laurie, Payton; Graduate, Biological Sciences, California State University, Chico, Presenting Author
Stachura, David; Faculty, Biological Sciences, California State University, Chico
Abstract: Hematopoiesis (blood development) is a complex and highly regulated process where hematopoietic stem and progenitor cells (HSPCs) differentiate into the numerous types of mature blood cells that make up the hematopoietic system for an organism’s entire life. Investigating and modulating the molecular pathways involved in HSPC formation and differentiation is important for treating a multitude of diseases including anemia, neutropenia, and leukemia. Due to the evolutionary conservation of the vertebrate hematopoietic system, we utilized zebrafish (Danio rerio) as a model organism. Three zebrafish stromal cell lines from known sites of hematopoiesis were generated that expand HSPCs. We performed RNA sequencing, comparing the transcript expression of these three stromal cell lines, and identified 447 genes that we believe are important regulatory factors in the hematopoietic system. A highly expressed transcript from these studies was chemokine (C-C motif) ligand 44 or ccl44. In order to test its effect on hematopoiesis we performed knockdown experiments using morpholinos (MO). Transgenic zebrafish lines with fluorescently labelled myeloid, erythroid, and lymphoid cells were injected with ccl44 MO and a decrease in those cell lineages was observed with flow cytometry, fluorescence microscopy, and quantitative PCR. Additionally, HSPC numbers were reduced as assessed by clonal in vitro assays. Co-injection of MO and ccl44 mRNA had a rescue effect, restoring mature blood cell numbers and hematopoietic gene expression to control levels. These data indicate that ccl44 is an important gene involved in blood production. Overexpression of ccl44 may have utility in expanding HSPCs, which would be useful for improving the outcomes of patients with diseases such as anemia and neutropenia or undergoing bone marrow transplants.
Poster #: 89
Campus: CSU Los Angeles
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: neural crest, CRISPR, zebrafish
Project Title: Creation of dyrk1b Mutants for Analysis of Potential Roles in Craniofacial Development
Author List:
Medina, Eric; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Thomas, Chad; Graduate, Biological Sciences, California State University, Los Angeles
Zitser, David; Undergraduate, Biological Sciences, California State University, Los Angeles
Ashour, Nadia; Undergraduate, Biological Sciences, California State University, Los Angeles
Nissen, Robert; Faculty, Biological Sciences, California State University, Los Angeles
Abstract: Infants born with cleft lip with or without cleft palate (CL/P) have a five-fold higher mortality rate during their first year. Wdr68 is a highly conserved scaffolding protein important for craniofacial development in vertebrates. Wdr68 lacks enzymatic activity but instead presents numerous surfaces to which over 100 protein interactions have been described. Two of the most well characterized binders to Wdr68 are members of the Dual-specificity Tyrosine-phosphorylation Regulated Kinase-1 (DYRK1) family, Dyrk1a and Dyrk1b. We created dyrk1a loss-of-function zebrafish mutants and found they lack craniofacial abnormalities (described elsewhere). Thus, Dyrk1b is the next candidate partner to Wdr68 for mediating craniofacial development. Abdominal Obesity-Metabolic Syndrome-3 (AOMS3) is caused by haploinsufficiency for human DYRK1B. However, a complete knockout of DYRK1B remains unexamined and may reveal more severe defects, including craniofacial defects. Here, we present progress using the single-guide RNA/Cas9 (sgRNA/Cas9) gene-editing system to produce dyrk1b loss-of-function zebrafish mutants. We successfully detected dyrk1b-sgRNA/Cas9 endonuclease-mediated cleavage of the dyrk1b target DNA region using an in vitro assay. We further detected dyrk1b genomic locus-specific cleavage in 6 of 6 Founder (F0) embryos injected with the dyrk1b-sgRNA/Cas9 gene-editing system. Thus, we have detected cleavage activity both in vitro and in vivo. Notably, these embryos showed various and multiple changes to the dyrk1b locus suggesting at least some genetic mosaicism in the F0 embryos. The DNA sequence of the dyrk1aa locus is highly similar to the dyrk1b locus but was found to be unaffected in 6 of 6 dyrk1b-sgRNA/Cas9-injected embryos suggesting a high degree of target specificity towards the dyrk1b locus. Several additional dyrk1b-sgRNA/Cas9-injected F0 embryos were raised to adulthood. At the time of this writing, one female F0 has been successfully outcrossed to wildtype zebrafish to produce F1 offspring potentially carrying germline dyrk1b mutations. At 3 months age, F1 animals will be tailfin genotyped to identify potential dyrk1b mutant carriers. Additional progress isolating and studying alleles will be presented. No other animal model for dyrk1b loss-of-function exists at the time of this writing. Thus, our results will lay the foundation for future studies of Dyrk1b-mediated activities for AOMS3 and craniofacial development. This work is supported by CSUPERB.
Poster #: 90
Campus: CSU Los Angeles
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Candida albicans, Transcriptome analysis, Metabolism
Project Title: Transcriptome analysis reveals that Yeast Casein Kinase 2 is involved in nutrient sensing and starvation response of Candida albicans.
Author List:
Liboro, Karl; Graduate, Biological Sciences , California State University, Los Angeles, Presenting Author, Eden Award Nominee
Abarca, Melvin; Undergraduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Park, Hyunsook; Faculty, Biological Sciences, California State University, Los Angeles
Abstract: Candida albicans is an opportunistic fungal pathogen that is a large source of morbidity and mortality, infecting women at least once in their lifetime as a yeast infection as well as being the fourth leading cause of bloodstream infections in the United States. Due to a lack of effective antifungals, new drug targets are required—Yeast Casein Kinase 2 (YCK2) is a conserved eukaryotic casein kinase family that has been shown to be important in C. albicans morphogenesis, a key virulence trait. Though exact mechanisms are unknown. To identify the metabolic pathways by which YCK2 governs, we evaluated the transcriptome of a YCK2 deletional mutant strain by RNA-seq analysis. Results suggest that the deletion of YCK2 disrupts glucose sensing, putting the YCK2 mutant into a perpetual mode of starvation. This leads to upregulation of genes related to starvation responses such as gluconeogenesis (through beta-oxidation and glyoxylate cycle) and hyphal formation (through cAMP-PKA pathway). Results also indicate that the Hog1-MAPK pathway is active, suggesting that the YCK2 mutant is under stress. Increased peroxisomal beta-oxidation produces hydrogen peroxide, a source of ROS that would explain the upregulation of Hog1-MAPK oxidative stress genes as well as the upregulation of arginine biosynthetic genes. However, ATP quantification revealed higher levels of ATP in the YCK2 mutant in most carbon sources. This contradicts the idea of starvation since gluconeogenesis is upregulated at low ATP levels when no high energy carbohydrates such as glucose are present. This suggests a mitochondrial dysfunction where perhaps acetyl carbons that would normally funnel from beta-oxidation to the glyoxylate cycle are instead funneling through the TCA cycle and into oxidative phosphorylation. A follow-up study with Methylene Blue (MB), a known inhibitor of cellular respiration and filamentation, showed that the YCK2 mutant was non-responsive to MB, suggesting that increased ATP production may be independent from the constitutive filamentation of the YCK2 mutant.
Poster #: 91
Campus: CSU San Marcos
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Saccharomyces cerevisiae, DNA replication, Repeat expansion diseases
Project Title: Contraction of Myotonic Dystrophy Type 2-Causing CCTG DNA Repeats
Author List:
Papp, David ; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Hernandez, Sophia; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Kim, Jane; Faculty, Biological Sciences, California State University San Marcos
Abstract: Expansions of simple DNA sequence repeats are the cause of over 30 genetic diseases, such as (CAG)n repeats in Huntington’s Disease, (CGG)n repeats in Fragile X syndrome, and (CCTG)n repeats in Myotonic Dystrophy Type 2 (DM2). DM2 is an inherited muscular disorder with no cure as of yet, that results in progressive muscle weakness, sight loss, and cardiac abnormalities. DM2 is caused by an expansion of CCTG DNA repeats in the ZNF9 gene, and affected individuals can have up to 5000 repeats at the locus. We used budding yeast S. cerevisiae to investigate genetic and environmental factors that contribute to CCTG repeat contractions, which may be therapeutically beneficial to target in DM2 patients. Expansions and contractions of trinucleotide (CTG, CAG, and CGG) repeats have been shown previously to be suppressed by the helicase Srs2, which can unwind secondary structures caused by these repetitive sequences. We utilized quantitative contraction assays and growth spot assays to observe the effect that knocking out Srs2 function would have on (CCTG)100 repeats and whether differences would be enhanced by additional environmental stress.
Results: We found that under normal conditions, knocking out Srs2 function did not have a significant effect on (CCTG)100 repeat contractions in a URA3 reporter gene, contrary to what was previously observed with trinucleotide repeats. In the presence of 1M sorbitol or 1M NaCl, which cause osmotic stress, we still found no significant difference in CCTG repeat contractions between wild type and srs2△ strains, though the salt treatment did significantly increase repeat contractions overall. Altogether, these studies demonstrate that the molecular mechanisms that influence the instability of different simple DNA sequence repeats are distinct and that environmental factors contribute to the instability of CCTG repeats. Additional studies are currently underway to identify other types of replication stress that increase CCTG contractions. This research was supported by CSUSM Summer Scholars to DP, NIH MARC GM008807 to SH, and NIH SC3 GM127198 to JCK.
Poster #: 92
Campus: Cal Poly San Luis Obispo
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: FXR-FGF19, pig, bile acid
Project Title: The farnesoid X receptor-fibroblast growth factor 19 (FXR-FGF19) axis is suppressed by prematurity and regulated by postnatal age in neonatal pigs
Author List:
Smith, Victoria; Undergraduate, Animal Science, California Polytechnic State University, San Luis Obispo, Presenting Author
Jiang, Yanjun; Department of Pediatrics, Baylor College of Medicine, Pediatrics
Thymann, Thomas; Comparative pediatrics and nutrition, University of Copenhagen, Pediatrics
Sangild, Per; Comparative pediatrics and nutrition, University of Copenhagen, Pediatrics
Maj, Magdalena ; Faculty, Biological Sciences, California Polytechnic State University, San Luis Obispo
Manjarin, Rodrigo; Faculty, Animal Science, California Polytechnic State University, San Luis Obispo
Burrin, Doug; Department of pediatrics, Baylor College of Medicine, Pediatrics
Abstract: Fibroblast growth factor 19 (FGF19) a key endocrine hormone in the bile acid production pathway. When digestion occurs, the liver secretes bile acids into the duodenum. When the bile acids are reabsorbed into the enterocytes of the ileum, they bind to the nuclear bile acid receptor, farnesoid X receptor (FXR), which triggers the secretion of FGF19 from the enterocyte into the portal circulation. FGF19 binds to receptors on the hepatocyte, which activates signaling pathways that inhibit hepatic bile acid synthesis by suppressing CYP7A1, the rate limiting enzyme in bile acid synthesis. Thus, the enterohepatic FXR-FGF19 axis acts as the main regulator of bile acid synthesis through a negative feedback mechanism. We hypothesized that prematurity would suppress the expression and activation of the intestinal FXR-FGF19 axis and would be increased with postnatal age. In this study piglets were delivered via cesarean-section at 10 days preterm and full term. The piglets were fitted with orogastric and umbilical arterial catheters and fed minimal enteral nutrition with bovine colostrum for 5 days and then fed bovine milk until 26 days of age. At days 0, 5, 11, and 26, tissue and plasma samples were collected. We developed a porcine FGF19 specific sandwich ELISA assay and used it to quantify peripheral plasma FGF19 concentrations. We also assayed distal ileum gene expression of FGF19, FXR, SHP, organic solute transporter alpha (OSTα), and ileal lipid binding protein (ILBP) by RT-qPCR. Results showed that plasma FGF19 levels are significantly lower in preterm vs. term newborn pigs. In addition, the activity of the FXR-FGF19 axis was marked altered by postnatal age; plasma FGF19 levels significantly increased 5 days after birth in preterm, but not term pigs. Between day 5 and day 26 of age, plasma FGF19 levels significantly decreased in both preterm and term pigs. Gene expression of intestinal FXR and FGF19 was lower (P<0.05) in preterm vs. term newborn pigs and decreased (P<0.05) between day 5 and day 26 of age. Expression of SHP, ILBP, and OSTα was also regulated by postnatal age. We conclude that the FXR-FGF19 axis is regulated by prematurity and postnatal age in neonatal pigs. Our findings are important for understanding bile acid homeostasis, FGF19 function, and growth of preterm and term infants in early postnatal life. Supported by National Institutes of Health Grant DK-094616 (D.G.B), USDA, and ARI-58982.
Poster #: 93
Campus: CSU Dominguez Hills
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: RNA-seq, evolution, motility
Project Title: Higher Expression of Motility Related Genes in Evolved Escherichia coli
Author List:
Henderson, Autumn; Graduate, Biology, California State University, Dominguez Hills, Presenting Author
Kram, Karin; Faculty, Biology, California State University, Dominguez Hills
Abstract: Understanding how Escherichia coli adapts to its environment helps to understand basic mechanisms of evolution, and may help develop more targeted treatments for infections. Incubating E. coli for long periods of time – days, weeks, even years without the addition of nutrients – allows cells to acquire mutations that are selected for in this stressful environment, ultimately leading to evolved populations that are better adapted to these conditions. What we do not know is exactly which genes or processes have changed in these adapted populations or what role these genes and processes play in the adaptation. We seek to identify genetic changes by determining differences in gene expression between unaged cells and cells aged for 10 days. The unaged and aged E. coli cells were each grown for four hours in LB broth, when growth rate is highest during the life cycle. Gene expression levels of the cells were then measured using RNA-sequencing. The RNA-seq data showed higher expression of genes related to motility (chemotaxis, flagellar, and motility protein genes) in aged cells. We hypothesized that the aged cells would be more motile and/or more chemotactic than the unaged cells. In order to assess whether aged cells are more motile than unaged cells we used motility assays to clearly visualize the movement differences between the unaged cells and aged cells. We found that aged cells were in fact more motile than unaged cells, supporting our hypothesis and confirming that gene expression data translated to an observable phenotype. We are currently performing chemotaxis assays to observe chemotactic sensing differences, and we predict we will see an increase in these behaviors in aged cells. In order to assess motility changes throughout the 10-day incubation period, we have identified target genes whose expression we can measure with qRT-PCR, and confirmed that these measurements match the RNA-seq data. Further, we are currently analyzing whole genome re-sequencing data in order to identify mutations that lead to the phenotypic changes seen here. Lastly, we are in the process of determining if motility plays a role in survival by competing non-motile strains with wild-type strains in long-term cultures. These data together will provide an overall picture of the role of motility in survival and adaptation.
Poster #: 94
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Lipoprotein Lipase, Glycolysis, Fatty Acid Metabolism
Project Title: Silencing the Lipoprotein Lipase Gene in Skeletal Muscle Cells Affects the Expression of Enzymes of Glycolysis and Fatty Acid Metabolism
Author List:
Mogul, Adam; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Pashanyan, Davit; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Medh, Jheem; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract: Background: The enzyme lipoprotein lipase (LPL) catalyzes the hydrolysis of triglycerides to free fatty acids and glycerol. In earlier reports, we demonstrated that reducing the expression of LPL in skeletal muscle cells increased insulin-stimulated glucose uptake, glycogen synthesis, and glucose oxidation but reduced oxidation of fatty acids to carbon dioxide. Thus, our data suggest that excessive expression of muscle LPL may precipitate insulin resistance. LPL may influence the oxidation of glucose by regulating the irreversible steps of glycolysis, namely, hexokinase II (HK 2) and phosphofructokinase (PFKM). Similarly, LPL may regulate fatty acid metabolism by affecting the transcription of carnitine palmitoyl transferase -1 (CPT1) and acetyl CoA carboxylase (ACC), rate-limiting steps of fatty acid β-oxidation and biosynthesis pathways.
Objective: To compare the expression of transcript and protein levels of the glycolytic enzymes HK 2 and PFKM in WT versus LPL-knockdown (LPL-KD) L6 rat muscle cells. To determine if silencing of the LPL gene in skeletal muscle cells affects the expression of CPT1 and ACC.
Methods: Lentiviral-mediated RNA interference was used by previous students to create a stable LPL-KD L6 cell line. Cells were treated with the antibiotic puromycin as a selection marker to ensure that only LPL-KD cells survived. Total RNA was isolated to make cDNA libraries. Specific primer pairs were used for real-time PCR amplification of LPL, HK 2, PFKM, CPT1, and ACC, along with β-actin as a housekeeping gene. Analysis of cellular protein concentration of HK and PFKM will be performed using western blotting (in progress).
Results: Quantitative PCR confirmed that LPL expression in the LPL-KD cells was <1% of that in WT L6 cells due to the shRNA silencing. Additionally, our data demonstrate that HK expression in the LPL-KD cells was an average of 2.5 fold higher than that of their WT counterparts. LPL-KD PFKM expression was on average about 70% of that in WT cells. Silencing the LPL gene also altered the transcription of CPT1 and ACC.
Conclusion: The knockdown of LPL in L6 cells increases gene expression of HK 2 while decreasing expression of PFKM. We hypothesize that increased HK expression facilitates glucose uptake into the cell. The finding that the glycolytic rate-limiting enzyme PFKM is repressed suggests that glucose-6-phosphate may be diverted to glycogen synthesis.
Acknowledgements: NIH Award SC3GM095413
Poster #: 95
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: (p)ppGpp, polyhydroxybutyrate, starvation
Project Title: Regulation of a dynamically-localizing polyester by three Caulobacter crescentus transcription factors
Author List:
Rocha, Alexa; Undergraduate, Biology, California State University, Northridge, Presenting Author
Schreiner, Shannon; USC
Stott, Kristina; JPL
Vu, Son; Olive View Hospital
Bueno, Diana; Pasadena City College
Arvizu, Ignacio; Staff, Biology, California State University, Northridge
Murray, Sean; Faculty, Biology, California State University, Northridge
Abstract: This investigation studies how the model organism Caulobacter crescentus responds to fatty acid starvation. To study this response, we created a FabH-depletion strain which induces fatty acid starvation. Starvation triggers the production of the intercellular signaling molecule (p)ppGpp in various bacterial species, including Caulobacter crescentus. (p)ppGpp 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). Immunoblot, transcriptional reporters, fluorescence microscopy, and mutant strains were used to create a model for PHB production during fatty acid starvation. (p)ppGpp is responsible for the expression of the transcription factor CtrA, which stimulates the synthesis of the transcription factor PhaR, which is necessary for PHB accumulation. PHB biosynthesis results from the efforts of three unique enzymes: PhaA, PhaB, and PhaC. We propose a model in which (p)ppGpp modulates phaC expression and the (p)ppGpp, CtrA and PhaR genetic pathway stimulates phaA and phaB expression. Our model illustrates how these three transcription factors work together to regulate expression of the PHB biosynthetic genes in response to fatty acid starvation.
This work was supported by NIH grant SC2 GM084860, NIH grant SC3 GM121234, and a CSUPERB Development grant to SM, NIH R25 GM063787 to MariaElena Zavala in support of AR, NIH R25 GM063787 to MariaElena Zavala in support of IA, and CSUN Interdisciplinary Research Institute in the Sciences Fellowships to KS and SS.
Poster #: 96
Campus: CSU Sacramento
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: gene amplification, Acinetobacter, siderophore
Project Title: Iron-limitation and siderophore formation increase cat gene amplification mutant frequencies in Acinetobacter baylyi
Author List:
Awwad, Habeeba; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Ghobashy, Rola; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Reams, Andrew; Faculty, Biological Sciences, California State University, Sacramento
Abstract: Gene amplification mutations are key contributors to cancer, pathogenesis, and the evolution of new genes. We aim to elucidate the regulatory and molecular mechanisms controlling these important mutations using a model system in the genetically tractable bacteria Acinetobacter baylyi. In this system, cells are placed onto minimum benzoate media agar plates that exclusively selects for mutant colonies carrying gene amplification of a chromosomal region encoding the cat genes involved in benzoate catabolism. Here, we show cat gene amplification mutant frequencies increase by over 50-fold when the cells are grown under iron-limiting conditions. Iron-limiting conditions are created by adding an iron chelator to the media, either 2,2’-bipyridine (BiP) or EDTA. We tested the effect of iron limitation on gene amplification frequencies under various conditions, including adding BiP to the non-selective growth media prior to selection, the selective minimum benzoate media, and both. Gene amplification mutant frequencies were highest (over 50-fold higher) when the iron chelator was added to both the pre-selective and selective growth conditions. Interestingly, we also show that under these iron-limiting conditions the cells secrete catechol, a toxic intermediate of benzoate catabolism, which has been previously shown to be a siderophore in A. baylyi. Our results suggest siderophore formation is required for cat gene amplification to detoxify the deadly accumulation of catechol by pumping catechol outside the cell. These studies were supported by the CSUPERB New Investigator Award.
Poster #: 97
Campus: Humboldt State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Genome Editing, Genetic Screens, Cell-Type Specific Promoters
Project Title: Development of Cell-Type Specific Cassettes for Stable Integration and Expression of Fluorescent Proteins and Gene Editing Tools in Human Neural Cells
Author List:
Johnes, Emma; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
De La Cruz, Francisco; Undergraduate, Biological Sciences, Humboldt State University
Anthoney, Kyle; Graduate, Biological Sciences, Humboldt State University
Steele, John; Faculty, Biological Sciences, Humboldt State University
Abstract: Tauopathies are a class of neurodegenerative disease that are associated with the toxic cytosolic accumulation of the protein tau in neurons, astrocytes, and/or oligodendrocytes. There are fourteen described tauopathies that include primary (e.g. corticobasal degeneration and progressive supranuclear palsy) and secondary (e.g. Alzheimer’s disease, Niemann-Pick disease type C1) tauopathies. Each disease is distinct in the region of the brain, cell type, and predominant isoform(s) of the tau protein that accumulates. Our lab’s goal is to understand how the autophagy pathway is utilized by these cell types to manage excess tau protein burden. However, in order to understand how each of these cell types regulates this pathway, we first needed to develop tools that will allow us to visualize these cell types and target genes in a cell-type specific manner. The goal of this project was to develop a suite of cassettes that act as cell-type specific reporters and can be used for facile cloning and expression of CRISPR gene editing or regulatory tools in the desired cell type.
In order to accomplish this goal, we developed a series of novel cassettes that: (i) can be integrated into the human AAVS1 locus by CRISPR gene editing and confer puromycin resistance; (ii) express an EGFP reporter driven by a cell-type specific promoter; (iii) contain a cleavable linker and unique cloning site for facile integration of CRISPR tools for genetic screens. We made unique cassettes with cell-type specific promoters to drive neuron-specific expression (SYN1, TUBA1A, or PRNP promoters), astrocyte-specific expression (GFAP promoter), oligodendrocyte-specific expression (CNP promoter), or expression in all mammalian cell types (EEF1A1). We successfully cloned, assembled, and screened plasmids for stable integration into the AAVS1 locus, then co-transfected plasmids along with dual nickase CRISPRs into HEK293t cells to confirm integration, puromycin resistance. As expected, we observed EGFP expression in HEK293t cells which integrated EEF1A1-promoter driven constructs, but not other cell-type specific constructs. Future studies will utilize these cassettes by integrating them into human induced pluripotent stem cell-derived neural progenitor cells use for genetic screens in our 3-D neural sphere cultures and disease models.
Poster #: 98
Campus: CSU Los Angeles
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: zebrafish, behavior, CRISPR
Project Title: Analysis of Dyrk1a in Zebrafish Development and Behavior
Author List:
Zitser, David; Undergraduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Ashour, Nadia; Undergraduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Godinez, Eduardo; Graduate, Biological Sciences, California State University, Los Angeles
Medina, Eric; Graduate, Biological Sciences, California State University, Los Angeles
Thomas, Chad; Graduate, Biological Sciences, California State University, Los Angeles
Sinclair, Jason; NIH
Burgess, Shawn; NIH
Nissen, Robert; Faculty, Biological Sciences, California State University, Los Angeles
Abstract: The Dual-specificity tyrosine phosphorylation-Regulated Kinase (Dyrk) gene family is a highly conserved group of protein kinases. Dyrk1a is implicated in various processes including neuronal proliferation, differentiation, and craniofacial development. While overexpression of Dyrk1a is associated with Down Syndrome and Alzheimer’s disease, Dyrk1a haploinsufficiency causes microcephaly in humans. While it is known that the mouse Dyrk1a-/- mutant is an early embryonic lethal, detailed studies of craniofacial development have not been completed in the mouse knockout model. Therefore, we are creating a zebrafish model for the study of Dyrk1a in early development. Notably, while human and mouse have only a single Dyrk1a gene, the zebrafish has two paralogous family members, dyrk1aa and dyrk1ab. Here, we present the creation and characterization of dyrk1aa and dyrk1ab mutants. Zebrafish founders were mutagenized by microinjection of sgRNAs and Cas9 mRNA, raised to adults, and outcrossed to wildtype. We then screened the resultant F1 animals by PCR to identify carriers for mutant alleles of dyrk1aa and dyrk1ab. We recovered seven different alleles for dyrk1aa and five different alleles for dyrk1ab. We focused our characterization of dyrk1aa on two alleles: dyrk1aacsu32 is a 4bp deletion causes a reading frame-shift and dyrk1aacsu31 is a 68bp deletion that removes a 3’-splice site. We focused our characterization of dyrk1ab on one allele: dyrk1abcsu40 is a 15bp deletion plus 2bp insertion that causes multiple splicing and reading frame-shifts. Surprisingly, the dyrk1aacsu32;dyrk1abcsu40 double mutants are viable and fertile suggesting divergence of Dyrk1a functions between zebrafish and mammals. Interestingly, we found preliminary evidence suggesting impaired social behaviors in the dyrk1aacsu32;dyrk1abcsu40 double mutants relative to wildtype zebrafish. Further progress on characterizing the mutants will also be presented. This research will improve our understanding of the functions of dyrk1aa and dyrk1ab in early development with potential implications for therapeutic interventions. This work was supported by a grant from the NIH and CSUPERB.
Poster #: 99
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila melanogaster, microbiome, evolution
Project Title: The evolutionary relationship between microbiome composition and the host, Drosophila melanogaster
Author List:
Hajy Heydary, Yasamin; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Courville, Robert; Graduate, Biological Science, California State University, Fullerton
Abstract: The interaction between the microbiome and host, and the effects the microbiome has on several physiological characteristics of the host such, as longevity, have been the object of intense inquiry in the past few years. However, the effect of host evolution for longevity differentiation on the microbiome composition has not been investigated in the past, which is the main purpose of this study. This research attempts to address whether the micobiome composition of long-lived and short-lived Drosophila melanogaster populations differ significantly. We analyzed metagenome data from 10 long-lived populations and 10 short-lived populations of D. melanogaster for differences in microbial DNA using Metaphlan. Populations with longer life spans had microbiome compositions dominated with the phylum Proteobacteria whereas the populations with shorter life spans have microbiomes dominated with the phylum Firmicutes. This result was surprising given that previous studies had shown that introducing Proteobacteria to the guts of flies made them short-lived. To confirm our metagenome results, we homogenized flies from 5 short-lived and 5 long-lived fly populations and plated the homogenates on MRS medium. The results supported our metagonome findings, with longer lived populations having more Proteobacteria and shorter lived populations having more Firmicutes. For future studies, the effects of the microbiome composition on host evolution for lifespan differentiation will be explored.
Poster #: 100
Campus: Humboldt State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords:
Project Title: Loss of Lgl1 affects MAPK and mTOR signaling and cellular migration and anchorage independent growth of murine neural progenitor-like cells
Author List:
Romero, Angelica; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
King, Megan; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
Gonzalez, Casiana; Undergraduate, Biological Sciences, Humboldt State University
LaCourse, Monique; Graduate, Biological Sciences, Humboldt State University
Otis, Sharon; Graduate, Biological Sciences, Humboldt State University
Alvarez, Christina; Undergraduate, Biological Sciences, Humboldt State University
Dombrowski, Matthew; Undergraduate, Biological Sciences, Humboldt State University
Sprowles, Amy; Faculty, Biological Sciences, Humboldt State University
Abstract: Lethal giant larvae is a tumor suppressor gene with roles in cell polarity and proliferation (Mechler et al. 1985; Lee et al. 2006; Humbert et al. 2003). Originally identified in a Drosophila genetic screen (Gateff 1978), mammalian phenotypes resulting from the loss of Lgl1 suggest the protein is involved in pathways associated with brain tumor formation. An abundance of neuroblastic rosette-like structures resembling human juvenile undifferentiated neuroectodermal tumors are found in the subventricular zone (SVZ) of Lgl1-/- embryonic mice (Klezovitch et al. 2004) and loss of the human ortholog of Lgl1, HUG1, is coincident with several aggressive human neoplasias including glioma (reviewed in Ellenbroek et al. 2012). To better understand how changes in intracellular signaling might affect the cancer properties associated with loss of Lgl1, we generated a Lgl1 -/- neural progenitor-like cell line from cells isolated from the SVZ of the Lgl1 flox/flox transgenic mice (Klezovitch et al. 2004). Immunoblot analyses show increased phosphorylation of p38 in Lgl1 -/- cells cultured as neurospheres and increased ERK and mTOR phosphorylation when cells are adhered to laminin coated coverslips. Lgl1-/ – and Lgl1 flox/flox cells were then evaluated for changes in cellular proliferation, migration, and anchorage independent growth in the presence of chemical inhibitors to p38, JNK, ERK, and mTOR. Our results show Lgl1-/- cells have increased migration rates that are sensitive to mTOR inhibition. Lgl1 -/- cells also exhibit changes in anchorage independent growth that are more sensitive to p38 inhibition. These results support previous work that signifies the importance of mTOR and MAPK signaling in cancer phenotypes and suggest potential new targets for the treatment of aggressive human brain cancers that do not express of HUG1.
Poster #: 101
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: cell cycle, neural crest, proliferation
Project Title: Analyzing cell cycle and proliferation during neural crest cell development in avian embryos
Author List:
Alfaro, Jocelyn; Undergraduate, Biology, California State University, Northridge, Presenting Author
Sugumar, Malavika; Undergraduate, Biology, California State University, Northridge, Presenting Author
Rogers, Crystal; Faculty, Biology, California State University, Northridge
Abstract: Neural crest (NC) cells play a crucial role in creating many adult cell types including craniofacial bone, pigment cells, and the peripheral nervous system. A population of premigratory NC cells form from the neural plate border and are specified in the dorsal neural tube. However, there is little known about how the small population of premigratory NC cells creates a population large enough to make all the necessary derivatives. With our current study, we analyze cell proliferation and the cell cycle in vivo during early neural crest cell formation and migration to determine when and where NC cells proliferate during early development. We hope to identify the important developmental time points that are crucial for NC cell division, migration, and differentiation. To clarify whether NC cells proliferate before, during, or after they migrate from the neural tube, we will analyze multiple developmental stages. We hypothesize that the NC cells divide prior to emigration and as they reach their final destinations, but that the cell cycle is halted during cell migration. To characterize these cells, we injected Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) in gastrula stage embryos and collected the embryos prior to NC specification, during the epithelial to mesenchymal transition (EMT), and during NC cell migration stages to determine the stages in which the ectodermal derivatives were progressing normally throughout the different stages of cell cycle. To verify the cell types being analyzed, we concurrently performed immunohistochemistry (IHC) on the embryos using markers for NC, neural tube, and non-neural ectoderm to locate the cells undergoing cell division at different stages of development. We found that after injecting the FUCCI and performing IHC, the cells that underwent division were primarily epithelial (premigratory) rather than mesenchymal (migratory). We plan to do further experimentation and analysis to understand the importance of timing and cell adhesion during these processes. Our project may create a new fundamental understanding of how NC cells behave in early stage embryos, which will increase our knowledge of defective developmental processes that may cause NC-related congenital defects such as craniofacial or pigment defects in vertebrate embryos.
Poster #: 102
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Regulatory T cells, Immunology , CRISPR
Project Title: Deubiquitinase Usp22 maintains Regulatory T cells identity by stabilizing Foxp3
Author List:
Prator, Grace; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author
Cortez, Jessica; University of California, San Francisco
Shifrut, Eric; University of California, San Francisco
Shaked, Oren; University of California, San Francisco
Marson, Alexander; University of California, San Francisco
Abstract: Regulatory T cells (Tregs) are a subset of CD4+ T cells that are vital to the maintenance of immune homeostasis. Tregs play a critical role in the suppression of inflammatory immune responses through the expression of the transcription factor Foxp3. Previous genetic lineage tracing studies demonstrated that Tregs can exhibit plasticity, where these ‘exTreg cells’ can acquire effector T cell phenotypes and are capable of secreting pro-inflammatory cytokines after losing Foxp3 expression. However, the molecular mechanisms that control Treg plasticity remain unclear. Using a pooled CRISPR screen approach in primary Tregs isolated from Foxp3-GFP lineage-tracing reporter mice, our lab has identified novel regulators that influence Foxp3 expression. Published work has shown that Foxp3 expression in Tregs can be regulated by deubiquitinating enzymes (DUBs) and ubiquitin ligases such as USP7 and Stub1, respectively. In this study, we sought to validate the role of previously undescribed candidate Foxp3 regulators including members of the SAGA deubiquitination module, Usp22 and Atxn7l3. We used CRISPR-Cas9 ribonucleoproteins (RNP) to knock out candidate genes in both human and mouse primary Tregs and identified changes in several Treg characteristic markers and pro-inflammatory cytokines by flow cytometry. We found that USP22 knockdown resulted in decreased in FOXP3, CTLA4, CD25, and IL-10 expression, but increased IFN-γ expression compared to a scrambled non-targeting control. This data suggests that USP22 could be playing an important role in maintaining FOXP3 expression and Treg identity. Investigating the plasticity of Tregs will allow us to create potential therapeutics targeting autoimmune diseases and cancer.
Poster #: 103
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: leukemia, cancer, GRB2
Project Title: Novel Antagonists of Growth Factor Receptor-bound Protein 2 (GRB2) Decrease Proliferation of Chronic Myeloid Leukemia (CML) Cells
Author List:
Aguiar, Stephanie; Graduate, Biological Sciences, California State University, Chico, Presenting Author
Griffin, Kallie; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author
Arpin, Carolynn; Faculty, Chemistry and Biochemistry, California State University, Chico
Stachura, David; Faculty, Biological Sciences, California State University, Chico
Abstract: Chronic myeloid leukemia (CML) is a disease that affects the normal growth of myeloid cells, blood cells that protect the body against foreign invaders, in the blood and bone marrow. In humans, 95% of CML cases are caused by a chromosomal translocation that inappropriately links the breakpoint cluster region (BCR) to Abelson murine leukemia viral oncogene-1 (ABL1), forming a mutant oncogene called BCR-ABL1. A protein that physically interacts with BCR-ABL1 is growth receptor bound protein-2 (GRB2), an intracellular adaptor protein involved in cell growth and differentiation. Specifically, BCR-ABL1 binds to a region of GRB2 known as the SRC homology-2 (SH2) domain. This interaction transforms hematopoietic stem and progenitor cells, initiating leukemic transformation. To prevent oncogenesis, we created four novel SH2 antagonists (NHD2-15, NHD2-92, NHD2-107, and NHD2-114) and tested their ability to prevent cell proliferation in the human BCR-ABL1+ K562 myelogenous leukemia cell line. The most significant growth reduction was observed 72 hours after the addition of 30uM of NHD2-15. Furthermore, adding drugs combinatorially (60uM NHD2-15, 30uM NHD2-114, and 1uM imatinib) to K562 cells showed over 2-fold growth reduction than with imatinib alone, which is the current frontline therapy for CML. Our previous studies indicate that NHD2-114 successfully bound to GRB2’s SH2 domain, but only minimally decreased cell proliferation on its own. However, NHD2-114 significantly reduced cell proliferation in our combinatorial assays; we are currently researching the molecular mechanism by which this occurs. While these drugs work together to efficiently kill K562 cells, we are also currently testing these drugs alone and in combination with each other on imatinib-resistant K562 cell lines that we created in the laboratory. Most patients become insensitive to imatinib treatment, so evaluating if these drugs can treat imatinib-resistant leukemia could lead to successful clinical treatments for people with this deadly disease.
Poster #: 104
Campus: CSU Fresno
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: thiols, transcriptomes, oxidative stress
Project Title: Transcriptome analysis demonstrates that low-molecular weight thiols have different functions in heterotrophic and autotrophic bacteria
Author List:
Thomas, Jason; Graduate, Biology, California State University, Fresno, Presenting Author, Eden Award Nominee
Rawat, Mamta; Faculty, Biology, California State University, Fresno
Abstract: Mechanisms to combat oxidative stress are critical for cell survival. Reactive oxygen species (ROS) resulting from a range of internal and external processes cause damage to critical cellular components including proteins, nucleic acids, and lipids. All bacteria must be able to withstand internally generated ROS that is formed from cell metabolism and aerobic respiration. Photosynthetic bacteria also produce ROS as byproducts of the photosynthesis. Pathogens also face external ROS released by an oxidative burst in neutrophils and macrophages. As a result, bacteria have developed multiple strategies to detoxify or prevent damage from these stressors, one of which is the production of low molecular weight (LMW) thiols that react directly with oxidants or donate reducing equivalents to enzymes that reduce oxidants. We have characterized mutants disrupted in the biosynthesis of these LMW thiols and shown that the lack of LMW thiols results in sensitivity to oxidative stress and attenuation of virulence in pathogens. To further delineate the role of LMW thiols in bacteria, we performed RNAseq analysis, followed by quantitative real time PCR on two pathogenic bacteria, which differ in their primary LMW thiol. Differential gene expression of LMW thiol mutants in Pseudomonas aeruginosa, which uses glutathione (GSH) as its main LMW thiol, and Staphylococcus aureus, which uses bacillithiol (BSH) as its main LMW thiol, was analyzed. We also examined two photosynthetic bacteria, Synechococcus PCC7942, which utilizes GSH, and Synechocystis PCC6803 which utilizes ergothioneine (ESH) as well as GSH. Our results indicate that genes involved in anaerobic respiration using nitrate as the final electron acceptor are induced in the pathogens containing structurally different LMW thiols. There is also a downregulation in genes related to toxin resistance and antibiotic detoxification supporting the attenuated phenotype of these mutants. In the cyanobacterial mutants, we did not see any induction of anaerobic respiration. Paradoxically, genes involved in photosynthetic pigment production were induced, suggesting an increase in photosynthesis. In addition, genes involved in NAD salvage pathways were downregulated, indicating a lesser need for this cofactor in the cyanobacterial mutants. This research provides further insight into the role of LMW thiols in bacteria and reveals similarities and differences in function of LMW thiols in disparate bacteria.
Poster #: 105
Campus: CSU Monterey Bay
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Genomics, Bioinformatics, Evolution
Project Title: The first draft de novo genome sequence for the hemiclonal fish, Poeciliopsis monacha
Author List:
Wildermuth, Katarina; Undergraduate, School of Natural Sciences, California State University, Monterey Bay, Presenting Author
Arbuckle, Talon; Northwest Indian College, Northwest Indian College
Johnson, Shannon; Monterey Bay Aquarium Research Institute
Vrijenhoek, Robert; Monterey Bay Aquarium Research Institute
Jue, Nathaniel; Faculty, School of Natural Science, California State University, Monterey Bay
Abstract: Poeciliopsis monacha is a small species of fish found in desert headwater springs of northwestern Mexico. Its habitat is known for fluctuating resources and temperatures, which has led to severe bottlenecking and localized extinction. As a result, P. monacha has evolved three forms of reproduction; gynogenesis, hybridogenesis, and sexual reproduction. Gynogenesis is a form of asexual reproduction in which sperm is needed to initiate development of the egg. Hybridogenesis is a type of hemiclonal reproduction in which a male from another species has genetic information represented in the immediate hybrid offspring but not in future generations. In comparison to other asexually reproducing vertebrates, P. monacha has higher levels of clonal variability that may be explained by multiple hybrid origins. A complete and annotated genome from all species would provide further insight, yet only a mitochondrial genome has been sequenced. We developed a project to produce a draft genome assembly using genomic and transcriptomic analysis. In this study, we present the first high-quality reference genome for a hybridogenetic reproducing P. monacha. The initial (1x) genome was generated using paired-end Illumina sequencing and assembled with PLANTANUS. Second (2x) and third (3x) scaffolds were created using the preceding sequence as a reference. The alignment of sequence reads was improved using BOWTIE2 read mapping, and the quantity of repetitive sequences was assessed using RepeatMasker. The 1x-, 2x-, and 3x- scaffold genomes were evaluated for quality and completeness using BUSCO and QUAST. A transcriptome was then assembled using the EviGene pipeline and used to train Augustus, a gene prediction tool. The predicted genomic and transcriptomic functional annotation results were compared using Blast2GO. Comparative analysis of Jukes Canter distance between similar species was used to examine rates of protein evolution. QUAST determined that the 3x-scaffold genome was the highest quality with an N50 value of 659,792, a length of 779,902,978 nucleotides and 75.1% mapped. It also had the lowest number of contigs. Our results suggest that the de novo 3x-scaffold genome is a high quality first draft sequence. Analysis of Jukes Canter distances indicated P. monacha is undergoing higher rates of genetic evolution in comparison to similar species. These results show that P. monacha may have greater genomic plasticity than originally predicted and merits further investigation.
Poster #: 106
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: apoptosis, survival, neural crest
Project Title: Cadherin-11 is required for the specification and cell survival of neural crest cells
Author List:
Manohar, Subrajaa; Graduate, Biology, California State University, Northridge, Presenting Author
Camacho, Alberto; University of Ottawa
Rogers, Crystal; Faculty, Biology, California State University, Northridge
Abstract: The neural crest (NC) is a multipotent embryonic population of stem-like cells that form various tissues in vertebrates including pigment cells, craniofacial bone and cartilage, and the peripheral nervous system. NC cells are ectoderm-derived progenitor cells that begin as epithelial cells in the neural tube, but detach and migrate throughout the body after undergoing an epithelial to mesenchymal transition (EMT). NC cells express a variety of cell adhesion molecules, including cadherin proteins, which control their specification, EMT, and migration. Abnormal development of NC cells can lead to multiple congenital defects as well as NC-derived cancers such as neuroblastoma and melanoma. Here, we identify the role of Cadherin-11 (CDH11) in early chicken NC development. CDH11 is a type II cadherin protein that is crucial for NC cell migration in amphibian embryos, and also regulates cell survival, proliferation, and migration in cancer cells. Using immunohistochemistry, we determined that CDH11 protein has previously unreported dynamic expression, which co-localizes with Sox2 in neural progenitor cells in early embryos. Then, as NC cells are specified in the dorsal neural tube and begin to undergo EMT, CDH11 becomes restricted to pre-migratory and migratory NC cells and is down regulated in the neural tube. Electroporation of a translation-blocking CDH11 morpholino at gastrula stages leads to a reduction of Pax7 and Sox9-positive NC cells in the dorsal neural tube, but has no effect on more ventral neural tube cells marked by Sox2, suggesting that CDH11 is required specifically for NC specification. We also determined that CDH11 is required for NC cell survival, as loss of CDH11 increases p53-mediated programmed-cell death, and blocking the p53 pathway can rescue the neural crest phenotype. Our findings demonstrate a new requirement for CDH11 in NC development, and may increase our understanding of early NC-related developmental defects. We have additionally identified that CDH11 is expressed in multiple cancer cell lines, and our future experiments will determine its requirement for the survival of these cells.
Poster #: 107
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Muscle spindle afferent, mechanotransduction, electrophysiology
Project Title: Effect of Piezo2 gain of function mutations on muscle spindle afferent sensitivity to stretch in adult mice
Author List:
Klier, Nikola; Undergraduate, Biological Sciences, San José State University, Presenting Author
Masri, Sameer; Undergraduate, Biological Sciences, San José State University, Presenting Author
Than, Kimberly; Graduate, Biological Sciences, San José State University
Ma, Shang; The Scripps Research Institute
Patapoutian, Ardem; The Scripps Research Institute
Wilkinson, Katherine A.; Faculty, Biological Sciences, San José State University
Abstract: Proprioception, the sense of one’s location in space, is necessary for complex motor tasks. The most significant proprioceptors are thought to be the group Ia and group II muscle spindle afferent (MSAs) sensory neurons that innervate the muscle spindle. These neurons communicate information about muscle length and movement through their firing frequency of action potentials. Mechanotransduction of muscle stretch to neural firing requires the opening of the mechanically activated non-selective cation channel Piezo2. The human disease, distal arthrogryposis type 5, is caused by a gain of function mutation to the Piezo2 channel. Patients experience proprioceptive deficits, which are consistent with Piezo2’s role in the MSAs. In this study, we hypothesized that this gain of function mutation of Piezo2 expressed in mouse MSAs would lead to an increase in the MSA response to stretch. We used an in vitro mouse muscle-nerve preparation to record MSA firing activity. Briefly, the extensor digitorum longus (EDL) muscle and innervating deep peroneal branch of the sciatic nerve were dissected and perfused in an oxygenated synthetic interstitial fluid tissue bath. An extracellular recording electrode was placed on the sciatic nerve and individual MSAs were identified by waveform. The EDL was subjected to a progressive series of 4s ramp-and-hold stretches increasing in length (2.5, 5, and 7.5% of resting length). Average instantaneous firing frequency was observed during the beginning (initial static time (IST), 0.5s into stretch) and end of stretch (final static time (FST), 3.5s into stretch). We found that stretch sensitivity was significantly lower in mice homozygous for the gain of function mutation of Piezo2 (n=10, IST 14.99+11.06 Hz, FST 10.99+8.90 Hz, p<.05) than in wild type (n=15, IST 28.73+11.828 Hz, FST 20.94+8.78 Hz, p<.05) mice and heterozygous mice (n=10, IST 26.64+16.12 Hz, FST 18.46+9.80 Hz, p<.05). Both heterozygous and homozygous mice were less likely to have MSA firing at resting length and showed instability of firing during stretch, suggesting deficits in MSA signaling were still present in the heterozygotes. Since Piezo2 is more likely to open in the mutant mice, there must be some compensation by the MSA to reduce firing during stretch. Future studies will investigate the mechanisms for these changes in MSA mechanotransduction.
Poster #: 108
Campus: Cal Poly Pomona
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: 3D cell culture, invasion , breast cancer
Project Title: Use of a 3D cell culture-based assay to study breast cancer cell invasion
Author List:
Lolarga, Jade; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Liu, Junjun; Faculty, Biological Sciences, California State Polytechnic University, Pomona
Abstract: Metastasis occurs when the cancer cells migrate away from the primary tumor into other tissues in the body, which is the main cause of death in most cancer patients. It is a multistep process, and the initial step is cancer cell invasion. Studying the behavior of cancer cells during invasion is important to understand the molecular mechanism underlying the regulation of metastasis. Most of the current invasion assays are two-dimensional (2D) and use a plastic chamber that contains a porous membrane, which is suspended over a larger well that contains media and/or chemo attractants. However, the 2D invasion assay limits the understanding of the behavior of the cancer cells during invasion in vivo, which occurs in a three dimensional (3D) way. The 3D tumor spheroid invasion assay provides data that closely represents the physiological behavior of cancer cells in vivo in response to therapeutic drugs and is therefore rapidly gaining popularity among cancer researchers. The hang drop method generates cell aggregates into spheres that are subsequently transferred to a 3D culture. This allows the cells to invade through the extracellular matrix (ECM) in a 3D culture setting, which is composed of Matrigel and type I collagen. Our study uses two breast cancer cell lines, BT549 Twist+ and BT549 Twist-, as models to study the regulation of breast cancer cell invasion in a 3D culture setting. BT549 is a triple negative breast cancer cell line that expresses a high level of Twist1. While Twist+ cells maintain the high level expression of Twist1, the Twist- cells have been stably transfected with a Twist1 shRNA, and the expression of Twist1 is largely suppressed. A time-lapse analysis of the cells in the 3D culture was done to monitor the invasive distance of each cell line for 72 hours. The results showed that in comparison to the BT549 Twist- cells, the BT549 Twist+ cells are far more aggressive and invasive based on the cell migration from the original sphere, which is consistent with our 2D invasion assay results. In conclusion, our 3D cell culture-based invasion assay is a great alternative to traditional 2D invasion assay and could potentially be used to study the effects of therapeutic drugs during the treatment of metastatic cancers.
Poster #: 109
Campus: CSU Fresno
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: speciation, genetics, experimental evolution
Project Title: The genetic architecture and tempo of hybrid fitness recovery
Author List:
Saini, Simran; Graduate, Biology, California State University, Fresno, Presenting Author
Ross, Joseph; Faculty, Biology, California State University, Fresno
Abstract: Relatively little is known regarding the process of species formation: how two very similar organisms begin to produce hybrid offspring that are either sterile or unviable. Such hybrid dysfunction arises because those offspring contain incompatible alleles of genes contributed by both parents at fertilization. This reduction of hybrid fitness is one of the major forces leading to speciation. Studies conducted on a nematode species, Caenorhabditis briggsae, have shown that hybrids formed from two different wild populations exhibit reduction in the fitness (fecundity) of the F1 generation itself. In many cases, this decrease has been observed to persist to the eleventh generation, suggesting that this genetic incompatibility might be of the type that would facilitate speciation. However, an increase in fitness has also rarely been seen at the eleventh generation. This finding raised the intriguing possibility that hybrid genetic incompatibilities that immediately decrease organismal fitness can be compensated in subsequent generations. This mechanism would be expected to reverse the speciation process. In order to understand the frequency and tempo of hybrid fitness recovery, we created and monitored the fitness of novel hybrid lines every generation for fifteen generations. Some of the lines exhibited a sudden fitness decrease and then gradual fitness recovery. Such a pattern of fitness change suggests the possibility that multiple compensatory mutations have occurred in these lines. The future goals of this research trajectory are to determine whether de novo mutations cause the observed fitness recovery and then to identify the molecular details of the fitness recovery process. Achieving this goal will help us better understand the process of speciation by studying mechanisms that oppose it.
Poster #: 110
Campus: Humboldt State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Plasmids, Genome editing, RNA knockdown
Project Title: Development of a novel tetracycline-inducible Cas13a toolset for targeted degradation of cellular RNAs
Author List:
Nisson, Haley; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
Perez Espinal, Maribel; Undergraduate, Biological Sciences, Humboldt State University
Pope, Amanda; Undergraduate, Biological Sciences, Humboldt State University
Rodriguez, Miranda; Undergraduate, Biological Sciences, Humboldt State University
Steele, John; Faculty, Biological Sciences, Humboldt State University
Abstract: RNA interference (RNAi) technology, a common method for knockdown of mRNA, offers the user limited specificity in targeting: only mRNA can be cleaved, and not all siRNA/shRNA cleave the target mRNA – resulting in off target effects which decrease accuracy of the cell line by altering non target mRNA. CRISPR/Cas9, zinc finger endonucleases, and TALENs are also commonly used to target genomic DNA in order to knock out or disrupt gene function. Alternatively, the CRISPR/Cas13a system is capable of specific RNA knockdown via complementary base pairing to an RNA guide (crRNA), increasing precision and accessibility from past RNAi technology. The goal of this project was to generate a cell line with stably integrated tetracycline-inducible Cas13a for use as a tool for studying disease state cells. In order to accomplish this goal, we designed, cloned fragments, and assembled two novel plasmids: pLizzard-crRNA and AAVS1-Cas13a-Repair template. The pLizzard-crRNA plasmid contains a U6 promoter for expression of non-coding RNAs, crRNA structure with a target sequence cloning site, and EF1alpha promoter driving expression of the reverse tetracycline transactivator (rtTA) and blasticidin resistance genes with a cleavable T2A linker. The AAVS1-Cas13a-Repair template contains the AAVS1-targeting homology arms, puromycin resistance gene, and TRE3G tetracycline response element containing promoter driving the fluorescent protein EGFP and Cas13a with a cleavable T2A linker. Both plasmids were constructed by Hifi assembly using PCR cloned DNA fragments, screened for proper assembly, then co-transfected into human embryonic kidney (HEK293t) cells. In the presence of the rtTA (encoded by pLizzard-crRNA) and doxycyline, cells expressed EGFP and Cas13a. We then used dual nickase CRISPRs to insert the AAVS1-Cas13a-Repair template into the AAVS1 locus of the human genome of the HEK293t cells and confirmed stable integration by puromycin selection. The next step in testing plasmids will be to test stably integrated cells with pLizzard-crRNA plasmid to ensure inducibility and target specificity. Now we have a stably integrated tetracycline-inducible tool that allows us to target any RNA in a cell (rRNA, tRNA, mRNA, etc) that can be used for functional genetics studies at the RNA level and for the study of regulation of RNA splice variants. Future studies will involve stable integration of these tools into human induced pluripotent stem cells for use in our cellular disease models.
Poster #: 111
Campus: San Francisco State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Sinorhizobium meliloti, bacterial genetics, atomic force microscopy
Project Title: Examining Bacterial Flagella with Atomic Force Microscopy
Author List:
Mars, Diana; Graduate, Chemistry and Biochemistry, San Francisco State University, Presenting Author
Karp, Donna Grace; Undergraduate, Biology, San Francisco State University
Anguiano, Anthony; Undergraduate, Engineering, San Francisco State University
Villalpando, Victor Emmanuel; Undergraduate, Engineering, San Francisco State University, Presenting Author
Azadi, Mojtaba; Faculty, Engineering, San Francisco State University
Chen, Joseph; Faculty, Biology, San Francisco State University
Abstract: We used atomic force microscopy (AFM), accompanied by relatively facile sample preparation, to examine bacterial cells at the nanometer scale and to determine whether genetic mutants produce flagella, whip-like structures that act as propellers and invisible by optical microscopy. Together with functional assessment of motility in semi-solid medium, results from AFM allowed us to verify that specific strains produce flagella that do not rotate, while other strains fail to synthesize any flagella. Verification of these phenotypes enables us to investigate the contribution of functional flagella to biofilm formation and host infection during symbiosis between the nitrogen-fixing bacterium Sinorhizobium meliloti and legume plants. A better understanding of factors that contribute to efficient symbiosis and nitrogen fixation can facilitate future improvements in agricultural output. This work was supported by funding from the National Science Foundation (NSF MRI Award No. 1626611) and the College of Science and Engineering (COSE) at San Francisco State University (Mini-Development Award from the Center for Computing for Life Sciences).
Poster #: 112
Campus: CSU Fresno
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Ambient Particulate Matter, ROS, Gene knockdown
Project Title: Understanding the Biological Mechanisms Behind ROS Production Caused by PM 2.5 Using siRNA Gene Silencing
Author List:
Raval, Kosha; Graduate, Chemistry, California State University, Fresno, Presenting Author
Kaur, Mandeep; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Dejean, Laurent; Faculty, Chemistry, California State University, Fresno
Abstract: Seven million people die each year from air pollution, according to the World Health Organization (WHO). When oxidative stress exceeds normal levels, the antioxidant system of any eukaryotic organism can become overwhelmed. This can lead to various adverse health effects in humans, including cardiovascular and pulmonary diseases. The risk for developing these deleterious conditions is heightened by increased exposure to particulate matter 2.5 (PM2.5), which is composed of solid particles and liquid droplets of transition metals and organic compounds 2.5µm or less in diameter. Having established a cytotoxic link between PM2.5 and reactive oxygen species (ROS), a form of oxidative stress, we now seek to understand the underlying biological mechanisms through which PM2.5 interacts with alveolar macrophage cells and produces excessive ROS. Studies have suggested that the up regulation of heme oxygenase-1 (HO-1) during oxidative stress plays an essential role in the regulation of intracellular ROS levels. Our study consists in optimizing a gene knockdown protocol for HO-1 in a rat alveolar macrophage cell model, NR8383, to investigate the potential involvement of this heme oxygenase in a PM2.5-induced ROS response. Having established an optimal protein concentration of 50µg to see distinct HO-1 bands in western blots, we are using a range of small-interfering RNA (siRNA) concentrations, from 10nM to 30nM, in conjunction with our positive control, zymosan, to understand their cytotoxicity effects. Zymosan, extracted from the cell wall of saccharomyces cerevisiae, is a known inflammatory agent which stimulates ROS production in NR8383. Repeated trials of western blotting have confirmed that 4 hour zymosan treatments increase the HO-1 protein levels in NR8383. Our aim is to confirm HO-1 knockdown through quantitative reverse transcription polymerase chain reaction (qrt-PCR) and western blotting. The cells with the HO-1 knockdown will finally be subjected to a DCF fluorescence microplate-based assay to monitor their PM2.5-induced ROS response. We hypothesize that we will observe enhanced ROS production in PM2.5 treated HO1-knockdown cells due to the lack of the protective enzyme, HO-1. Understanding this biological mechanism through which PM2.5 is able to induce ROS will help us understand exactly how pollution is pernicious and advocate for global reduction of PM2.5 exposure to the world’s current 95% population that is overexposed to it.
Poster #: 113
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: XoxF, Methylobacterium extorquens, Rare Earth elements
Project Title: Identification and Characterization of Rare-Earth Transport in Methylobacterium extorquens
Author List:
Ayala, Elena; Undergraduate, Biological Sciences, San José State University, Presenting Author
Cai, James; Undergraduate, Biological Sciences, San José State University, Presenting Author
Vu, Huong ; Undergraduate, Biological Sciences, San José State University
Subuyuj, Gabriel; Undergraduate, Biological Sciences, San José State University
Wingett, Justin; Graduate, Biological Sciences, San José State University
Lien, Nicholas ; Undergraduate, Biological Sciences, San José State University
Skovran, Elizabeth; Faculty, Biological Sciences, San José State University
Abstract: Methylobacterium extorquens is a model organism for the study of methylotrophy, which is the ability to utilize single carbon chemicals as a sole source of carbon and energy. Recently, it was discovered that in addition to the Ca-dependent MxaFI methanol dehydrogenase, M. extorquens has a rare earth element (REE)-dependent methanol dehydrogenase, XoxF, which is preferentially expressed over mxaFI if REE are present in the environment. We have previously demonstrated that M. extorquens is capable of obtaining REE from electronic waste and mining ores enabling the development of a biological platform to recycle or mine for REE. Currently nothing is known about how REE are obtained or transported into the cell. To identify genes required for REE-dependent methanol oxidation, a transposon mutant hunt was performed which identified ~40 genes required for REE-dependent growth including a putative TonB-dependent receptor (TbdR) and ABC transporter. Mutant strains were reconstructed, and growth curve analysis indicated that the TdbR and ABC transporter are required for REE-dependent growth. Consistent with these findings, loss of the receptor and transporter components significantly reduced REE uptake from the growth medium and transcriptional reporter fusions showed that expression of xoxF was repressed in the mutant strains. However, after 120-250 hr, growth of the transporter mutants occurred due to adaption or second site suppression. During this growth lag expression of xoxF increased 2-fold, suggesting that trace amounts of REE are able to enter the cell and initiate REE-dependent gene expression. Additionally, once growth occurs, expression of xoxF was upregulated ~25-fold further. These results suggest that once a threshold of REE is obtained, xoxF expression is triggered and REE-dependent methanol oxidation and growth can occur. Future work will focus on identifying the mechanism by which insoluble REE are acquired and the secondary mechanism responsible for REE uptake in the absence of the main REE transport machinery. Preliminary targets identified through genetic studies and comparative genomics include an outer membrane porin protein, the ferric citrate transporter, and a second ABC transporter. Results from this work will help engineer M. extorquens as a more efficient platform for REE recycling while uncovering fundamental mechanisms in REE metabolism. This work was funded by a CSUPERB Joint Venture grant and an NSF RISE fellowship.
Poster #: 114
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: gene regulation, promoter, cancer
Project Title: PROMOTER REGION ISOLATION AND CHARACTERIZATION OF SMALL LYMPHOCYTIC LYMPHOMA ASSICATED UVRAG
Author List:
Antikyan, Mary; Undergraduate, Biology, California State University, Northridge, Presenting Author
Billoo, Rabia; Undergraduate, Biology, California State University, Northridge, Presenting Author
Hain, Samantha; Graduate, Biology, California State University, Northridge
Malone, Cindy; Faculty, Biology, California State University, Northridge
Abstract: Mantle cell lymphoma is a more aggressive lymphocytic cancer with a short survival time, while small lymphocytic lymphoma is more a passive form of lymphocytic cancer with a longer survival time. The gene, UVRAG (UV Radiation Resistance Associated), was found to express differently in small lymphocytic lymphoma compared to mantle cell lymphoma via suppression subtractive hybridization. UVRAG is a protein-coding gene. Promoter characterization of this gene will allow us to identify the regulatory elements driving transcription as well as the evolutionary conservation of transcription factors affecting optimal transcription. The UVRAG promoter region was cloned into the pGC-Blue vector, then sub-cloned into the luciferase expression vector, pGL3 Basic. Deletion constructs of the recombinant plasmid were pre-determined by PCR by exclusion and transfected into Human Embryonic Kidney Cells. A total of six deletion constructs were designed, one of which was a natural cut site using SmaI. Transformed cells were grown on agar plates with ampicillin, DNA was purified and used to perform transfections. Harvested cells were then used to determine luciferase expression with a luciferase assay. Through luciferase data, it was apparent that a possible suppressor was found between the two smallest deletion constructs due to a large decrease in luciferase expression. A deeper look into these two deletion constructs will allow for the characterization of the UVRAG promoter and possibly allow for further deletion constructs and a better understanding of the expression of UVRAG.
Poster #: 115
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: cancer stem cell, acquired therapy resistance, single-cell protein methods
Project Title: PEAK1-Dependent Factors from Tumor Stroma Drive Tumor Growth and Therapy Resistance via Enrichment of Stem-Like Breast Cancer Cells
Author List:
Güth, Robert; Postdoc, Biology, California State University, Northridge, Presenting Author
Hamalian, Sarkis; Graduate, Biology, California State University, Northridge
Zervantonakis, Ioannis; Harvard Medical School
Lin, Jia-Ren; Harvard Medical School
Agajanian, Megan; Graduate, Biology, California State University, Northridge
Molnar, Justin; Graduate, Biology, California State University, Northridge
Geller, Cameron; Graduate, Biology, California State University, Northridge
Shiang, Sarah; Staff, Biology, California State University, Northridge
Sorger, Peter; Harvard Medical School
Brugge, Joan; Harvard Medical School
Abstract: Breast cancer is a highly prevalent disease that exhibits extensive cellular and molecular plasticity within and between patient tumors. These properties greatly diminish the effectiveness of existing treatments and often result in tumor recurrence following treatment. The signaling kinase PEAK1 is increased in breast cancer compared to normal mammary cells and promotes cancer cell plasticity, metastasis, and therapy resistance. Recently, PEAK1 has been found to be significantly increased in the stromal cell compartment of breast tumors compared to normal breast. Therefore, we hypothesized that PEAK1 expression in the tumor stroma may contribute to tumor progression and therapy resistance. We report here that elevated stromal expression of PEAK1 is associated with breast cancer recurrence in patients. We further demonstrate that PEAK1 expression within the stromal compartment occurs in cancer-associated fibroblasts (CAFs) and mesenchymal stroma cells (MSCs). Co-transplanting CAFs or MSCs with HER2-positive and ER-positive subtypes of breast cancer cells into chicken embryos resulted in increased primary tumor mass and promoted tumor resistance against targeted therapies. Notably, PEAK1 expression in MSCs was necessary for these effects, as silencing of PEAK1 within these cells abolished the protective effects exerted by MSC-conditioned media or MSC co-culture on breast cancer cells. To identify the molecular mechanisms that are responsible for the therapy resistance effect in this system, we used multiplexed immunofluorescence (CycIF) imaging to simultaneously track multiple protein markers at single-cell resolution. These studies showed that PEAK1 expression in MSCs is required for the survival of stem-like SOX2-positive breast cancer cells that remain high in p65-NFkB survival signaling activity following treatment with targeted therapies. Finally, to identify stromal factors initiating these effects, we employed protein array screens. These screens revealed that compared to control MSCs, PEAK1-silenced MSCs reduce expression of numerous protein factors that have been associated with tumor promoting functions. Analyses of clinical databases supports the directed targeting of these factors as a means to sensitize breast cancer cells to existing therapies, thus suggesting new strategies for improving breast cancer patient outcomes. Funding for this work was provided by NIH-NIGMS, CSUPERB, the Sidney Stern Memorial Trust, and Dr. Gary and Vera Sutter.
Poster #: 116
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: zebrafish, fecundity, embryogenesis
Project Title: Effects of Different Wavelengths of Light on Zebrafish Fecundity
Author List:
Cajias, Illiana; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author
Maciel, Sarah; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author
Belmonte, Rebecca; Graduate, Biological Sciences, California State University, Chico
Thapa, Samrat; Chico High School
Aggio, Julian; Undergraduate, Biological Sciences, California State University, Chico
Wolf, Anja; Undergraduate, Biological Sciences, California State University, Chico
Smith, Jesse; Graduate, Biological Sciences, California State University, Chico
Stachura, David; Faculty, Biological Sciences, California State University, Chico
Abstract: Danio rerio (zebrafish) have rapidly gained popularity in the study of vertebrate development and gene function. Zebrafish are an excellent model to study because of their fecundity, external embryo development, and transparency during early development. This allows scientists to easily perform large-scale genetic and drug screens and then observe the effects on a living, developing organism, which is not possible with mammals. Because large numbers of embryos are required to perform these experiments, we investigated ways to efficiently increase zebrafish embryo production while not being overly invasive or using chemicals. Therefore, we investigated how different wavelengths of visible light would affect zebrafish embryo production. To perform these experiments, we mated wild type fish while exposing them to different wavelengths of light. Fish were exposed to 12hrs of no light, followed by 5hrs with different colored light treatments. Once this light cycle was over, we collected and enumerated embryo production. Results indicated that fish exposed to blue and red light produced the most embryos, while fish exposed to white and green light produced the fewest. Although the exact mechanism behind increased embryo production is unclear, embryo generation is clearly altered by exposure to different wavelengths of light. These data will allow scientists to more efficiently increase embryo production without having to use chemicals or change their light/dark schedule. In turn, this will allow for further studies of the circadian rhythm of vertebrates, elucidate the effect of light exposure on molecular processes, and increase embryo production, which will make zebrafish an even better model organism for studying a multitude of biological processes.
Poster #: 117
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: PHB, formate dehydrogenase, Methylobacterium extorquens
Project Title: Transcriptional regulation of of the formate branch point and poly-hydroxybutyrate production in Methylobacterium extorquens
Author List:
Nguyen, Hoang; Graduate, Biological Sciences, San José State University, Presenting Author
Luu, Kim; Graduate, Biological Sciences, San José State University, Presenting Author
Raghuraman, Charumathi; Graduate, Biological Sciences, San José State University
Good, Nathan; Michigan State University
Martinez-Gomez, N. Cecilia; Michigan State University
Skovran, Elizabeth; Faculty, Biological Sciences, San José State University
Abstract: Methylobacterium extorquens is a model organism for understanding methylotrophy and is used to produce value added chemicals like bioplastics (PHB) from inexpensive feedstocks like methanol and formate. NAD(P)H plays a key role in the production of PHB and other value-added chemicals and is essential for energy generation and biosynthesis. However, the regulatory network that controls production of these reducing equivalents is unknown. The formate branch point is a crucial metabolic point as it governs the split between carbon assimilation and dissimilation. At this point, formate can be oxidized to CO2 by four distinct formate dehydrogenase enzymes (Fdh1,2,3,4) to provide energy and reducing power in the form of electrons (Fdh3) or NADH (Fdh1,2); or it can be converted to methylene H4F for assimilation via formate H4F ligase (FtfL). The transcriptional regulators controlling expression of each of the above genes is unknown. Here we show that two LysR-type transcriptional regulators, QscR and CosR, play a key role in the regulation of this branch point. We demonstrate that CosR is required for nucleotide homeostasis as loss of cosR results in 8.5-fold decreased NAD(P)H levels and accumulation of PHB. We hypothesize that nucleotide pools may be out of balance due to altered expression of the formate branch point genes. QscR is known to regulate the Serine Cycle in M. extorquens but previous microarray experiments suggested QscR may regulate the formate branch point genes as well. Using transcriptional reporter fusion assays, we show that qscR is required for activation of fdh1,2, and ftfL, and repression of fdh4 while cosR is required for activation of fdh1,4, and ftfL, and repression of fdh2,3. However, these assays do not determine if regulator function is direct or indirect. To determine direct interactions, gel mobility shift assays were performed using purified regulators and showed that both CosR and QscR bind to the fdh4 promoter region while QscR also binds to the fdh2 promoter. Our work identifies two transcriptional regulators that control a key branch point in methylotrophic metabolism which impacts reducing power and energy generation for the cell. A better understanding of the role of these LysR-type regulators may lead to increased PHB production for industry while increasing our knowledge about essential regulators for methylotrophy. Funding for this project was provided by San Jose State University.
Poster #: 118
Campus: CSU Northridge
Poster Category: Other
Keywords: Antimicrobial Peptides, Population Dynamics, Inoculum Effect
Project Title: Population Dynamics of Antimicrobial Peptides and Bacteria
Author List:
Talledo, John Paul; Graduate, Physics and Astronomy , California State University, Northridge, Presenting Author, Eden Award Nominee
Snoussi, Mehdi; Undergraduate, Biology, California State University, Northridge
Del Rosario, Nathan; Undergraduate, Biology, California State University, Northridge
Ha, Bae-Yeun; Department of Physics and Astronomy, University of Waterloo, Ontario, Waterloo, ON, Canada
Košmrlj, Andrej; Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ, USA
Taheri-Araghi, Sattar; Faculty, Physics and Astronomy, California State University, Northridge
Abstract: Antimicrobial peptides (AMPs) are broad-spectrum antibiotics that utilize electrostatics to target bacteria selectively. Like most antibiotics, AMPs need a minimum concentration to inhibit the growth of a bacterial population. Despite our knowledge of the molecular structures and membrane interactions of AMPs, we poorly understand AMP’s dynamics at the cellular and the population level. Here we demonstrate that the minimum inhibitory concentration (MIC) of AMPs is strongly dependent on the cell density, even in dilute cultures where direct cell-to-cell interactions are minimal. We hypothesize that this dependence arises because individual cell absorbs a significant number of AMPs which considerably reduces the effective concentration of AMPs in the culture. To investigate this hypothesis, we used a live single-cell imaging platform to track fluorescently tagged AMPs and the time evolution of their translocation into bacteria. We also developed a chemical reaction-like model to elucidate the kinetics of interacting bacteria and AMPs. Our single-cell analysis shows that bacteria not only absorb a significant fraction of AMPs from the culture but also retain them even after cell death, which sequesters AMP’s availability for attacking more cells.
Poster #: 119
Campus: CSU Fresno
Poster Category: Other
Keywords: Inquiry, Laboratory, Learning
Project Title: A novel and engaging approach to Introductory Chemistry Laboratories: A Zoo Inquiry Project
Author List:
Kamitono, Jordyn; Undergraduate, Biology, California State University, Fresno, Presenting Author
Donnelly, Dermot; Faculty, Chemistry, California State University, Fresno
Person, Eric; Faculty, Chemistry, California State University, Fresno
Abstract: The engagement of students within Introductory Chemistry Laboratories is often constrained by recipe-style structures that limit student decision-making and creativity. Laboratory structures should greater align with the experience of practicing scientists by providing students with an authentic investigative context and ownership in experimental practices. This mixed-method investigation analyzes the learning outcomes and perceived ownership of students within existing laboratory structures for an introductory chemistry course compared to a novel laboratory structure that utilizes zoo-inquiry projects (ZIPs). Students in the ZIP condition collect water samples from aquatic exhibits in the local zoo and work with these water samples throughout the semester to investigate topics such as salinity, pH, and thermodynamics. In groups, students explore these concepts by generating their own hypotheses, creating their own experimental design, and sharing their findings with their peers. The quantitative pre/post data for this study involves eight conceptual and experimental practice items developed and scored using the Knowledge Integration framework. The qualitative data for students’ science ownership was assessed through a post-course questionnaire. Analysis of the quantitative data shows that the ZIP treatment (n=141) and existing laboratory treatment (n=184) had significant conceptual and experimental practice gains while the qualitative data indicates that students within the ZIP treatment express greater ownership of investigations and less instructor dependence. These findings point to the important role of laboratory structure and context in enhancing independent and critical thinking in Chemistry learning.
Poster #: 120
Campus: CSU Fresno
Poster Category: Other
Keywords: soundscape, bats, biodiversity
Project Title: Using ultrasonic monitoring and acoustic analyses to detect and monitor bat biodiversity in Sequoia and Kings Canyon National Parks
Author List:
Seward, Robert; Undergraduate, Biology, California State University, Fresno, Presenting Author
Reece, Joshua; Faculty, Biology, California State University, Fresno
Abstract: The Sequoia and Kings Canyon National Parks are large protected areas that span an elevational gradient of 100 m to more than 3,000 m. These parks include habitat for countless species and provide ecosystem services such as clean air and water for millions of people. Bats are among the most diverse groups of mammals, and they are important both for the ecosystems that they inhabit and the humans that depend on those ecosystems. Because bats are nocturnal, small and difficult to detect, they are often not well surveyed. In Sequoia and Kings Canyon National Parks, there has been virtually no thorough assessment of what species of bats are present and where. Traditional bat monitoring efforts include mist-netting, which is when relatively short nets up to 30 meters in length and 3-5 meters high are stretched out during dusk. Bats fail to perceive the fine mesh of the nets and fly into them, becoming entangled; researchers free them, identify and measure them and then safely release them back into the environment. Modern advances in biotechnology have made it possible to deploy ultrasonic microphones and recorders that can record the ultrasonic echolocations that bats emit to navigate and hunt. These recordings can be analyzed to identify species of bats based on their species-specific echolocation calls. During the Summer and Fall of 2018 we conducted 16 nights of paired mist-netting and acoustic recording in Sequoia and Kings Canyon to compare the sampling results and determine the suitability of mist-netting relative to acoustic monitoring for assessing species diversity. We caught 6 species of bats in mist nets and recorded 16 species using the acoustic recorders. Our results suggest that acoustic recorders are a less time intensive and more accurate approach to surveying bat biodiversity. Learning how to identify recordings to species has the ancillary benefits of exposing students to analytical thinking, computer programing, and statistics.
Poster #: 121
Campus: CSU Fullerton
Poster Category: Other
Keywords: Heavy Metals, Electronic Cigarettes, Aerosol
Project Title: Quantification of the transfer mechanism of potentially harmful heavy metals to the inhaled aerosol particles generated by an electronic cigarette
Author List:
Rastian, Brian; Graduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Curtis, Daniel; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Abstract: Electronic cigarettes (e-cigarettes) are increasing in popularity despite uncertainties about their health effects. The commonly held belief among consumers is that e-cigarettes are a safer alternative to cigarettes. However, recent studies have shown that e-cigarettes may be a source of toxic heavy metal exposure. Importantly, the source of metals in the inhaled aerosol particles is not well understood, nor is the mechanism by which metals are transferred from the metal parts of the e-cigarette into the aerosol plume inhaled by the user. The purpose of this study was to quantify heavy metals systematically during the simulated use of an e-cigarette in order to better determine how metals are transferred from the e-cigarette into the inhaled aerosol plume. Ultimately, the goal of the study is to better understand the potential health risks of e-cigarettes.
E-cigarettes generate an aerosol particle plume by the vaporization and re-condensation of a liquid mixture (e-liquid) of propylene glycol, glycerol, nicotine, and flavorings, that is stored in a tank on the device. The vaporization of the e-liquid takes place by resistive heating of a metallic coil. In order to probe heavy metal transfer to the e-liquid in the storage tank and to the aerosol, this study measured the concentration of nickel and chromium in both the storage tank and in the collected aerosol plume while puffing the e-cigarette. Samples were collected every 10 puffs, up to 50 puffs, to determine if there was an increase in metal concentration the more times the e-cigarette coil was fired. Nickel and chromium were chosen due to their serious health effects which include respiratory disease and lung cancer. Concentrations were measured using graphite furnace atomic absorption spectroscopy.
In this study, a sharp increase in the chromium and nickel concentrations in both the storage tank e-liquid and in the aerosol particles was observed the more times the e-cigarette was fired during puffing. A control experiment indicated that firing the e-cigarette was required to transfer metals into the e-liquid. The results of this study indicate that nickel and chromium are first transferred into the storage tank e-liquid due to heating of the metal coil in an e-cigarette. Subsequent vaporization of the e-liquid transfers metals into the aerosol plume that is inhaled by the user.
Poster #: 122
Campus: CSU Bakersfield
Poster Category: Other
Keywords: sucidal ideation, neural networks, natural language processing
Project Title: A suicide ideation therapist-chatbot using entity and parts of speech co-reference model
Author List:
Manahan, Jose Luis; Undergraduate, CEECS, California State University, Bakersfield, Presenting Author
Cruz, Alberto; Faculty, CEECS, California State University, Bakersfield
Abstract: 10 million U.S. adults have had at least one major depressive episode. Most are not even aware of their condition and in the worst case it may be too much to bear. Despite professional services, suicide ideation continues to be a problem. We explore the potential of artificial intelligence to provide therapy via interactive chatbots. We anticipate this to be an effective modality with those who fear social stigmatization. The implementation of current methods, cognitive behavioral therapy and dialectical behavior theory (DBT), have restrictive interaction or force the interaction to consist of canned statements. We propose an approach that identifies the object or idea that is causing an individual’s distress. Previous approaches have attempted this, but were limited to nouns. We are the first to consider all parts of speech. Our chatbot was implemented in Python 3 with the libraries from Natural Language Tool Kit and SentiWordNet. A master character tree is trained through a large, publicly available corpus. A graph is developed mapping the relationship between each part of speech and multiple mini neural networks estimate the sentiment of arc to identify the source of the distress. The sentiment values can be used to improve DBT treatment methods. The neural nets have an absolute error of 0.1 to 0.2% and the accuracy of the association rates of 74.1 +/- 5.0%. We identified the inclusion of nonstandard phrases to be a source of error and this will be the focus of future work.
IRB Statement: We use publicly available data thus this work does not constitute as human subject research.
Poster #: 123
Campus: Cal Poly San Luis Obispo
Poster Category: Other
Keywords: fermentation, kinetics, engineering
Project Title: Investigation of the kinetics of continuous fermentation
Author List:
Koen, Nadine; Undergraduate, Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, Presenting Author
Butler, Renee; Undergraduate, Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, Presenting Author
Abstract: Brewing has been practiced for thousands of years over many cultures. Most brewed liquids, whether made commercially or at home, are produced in a batch fashion. Despite the potential advantages offered by continuous reactors, beer brewing, with one former notable exception, takes place in batch reactors even on the largest scales. Continuous fermentation offers the potential for decreased production time, labor costs and more control of the reaction conditions on the industrial scale. In the laboratory, from the perspective of kinetic analysis, continuous fermentation at steady-state may provide simpler calculations through algebraic, rather than differential, equations. We are exploring beer fermentation in a cascade of continuous stirred-tank reactors (CSTRs) utilizing commercially available free flocculent yeast. We are investigating the conditions necessary for steady-state operation at different flowrates and temperatures through a suite of analytical instruments: refractometer, hemocytometer, GC, and HPLC. Through our measured concentrations of active yeast, total fermentable sugar, ethanol and diacetyl (a noted flavor defect), we determine the rate constants associated with a set of coupled Monod-type empirical model equations. Our results to date have exhibited that it is possible to attain steady-state behavior in this type of reactor network without immobilization of the yeast. Our resultant kinetics models are shown to be robust enough to predict the steady-state concentrations and also the observed oscillatory behavior as the reactor system approaches steady-state. Future work includes using optimization algorithms to predict the reaction conditions in the CSTRs that will maximize the conversion to products and minimize the formation of undesirable components.
Poster #: 124
Campus: CSU Sacramento
Poster Category: Other
Keywords: Neurodevelopment, Drosophila melanogaster, Autism
Project Title: Developmental Exposure to Bisphenol-A Causes Axon Outgrowth Defects in Drosophila melanogaster
Author List:
Tinsley, Brendan; Graduate, Biological Sciences, California State University, Sacramento, Presenting Author
Nguyen, Uyen; Graduate, Biological Sciences, California State University, Sacramento, Presenting Author
Casiquin, Joyce; Undergraduate, Biological Sciences, California State University, Sacramento
Ceballos, Alex; Undergraduate, Biological Sciences, California State University, Sacramento
Danziger, Kaitlin; Undergraduate, Biological Sciences, California State University, Sacramento
Palacios, Yomira; Undergraduate, Biological Sciences, California State University, Sacramento
Sen, Yen; Undergraduate, Biological Sciences, California State University, Sacramento
Welch, Chloe; Undergraduate, Biological Sciences, California State University, Sacramento
Mulligan, Kimberly ; Faculty, Biological Sciences, California State University, Sacramento
Abstract: BACKGROUND & RATIONALE: Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders that affects 1 in 59 children in the United States. ASD has complex multifactorial etiologies, including genes and environmental factors. While hundreds of genes have been linked to ASD, much less is known about what environmental factors impact ASD pathophysiology. The gene x environment hypothesis posits that many environmental factors may only confer risk of ASD when combined with ASD risk genes. Thus, identification of environmental factors that confer risk of ASD and examination of how they interact with genetic risk factors is critical to improve our understanding of ASD etiology and to enact preventative measures. The overarching goal of this project is to examine the environmental chemical bisphenol-A (BPA) in combination with the ASD risk gene fragile X mental retardation 1 (fmr1), the most common single gene cause of ASD. The endocrine-disrupting chemical BPA is a high-production chemical commonly used in the production of plastics and epoxy resins; it is found in a vast number of products ranging from baby bottles to thermal receipt paper. The structure of BPA allows it to easily cross the placental and fetal blood brain barriers. Recent studies indicate that BPA affects neurodevelopmental phenotypes implicated in ASD, like neural stem cell proliferation and synapse formation. However, axon outgrowth—another critical neurodevelopmental phenotype associated with ASD—has not been well explored. In addition, there are no studies examining how BPA interacts with ASD risk genes. METHODS & RESULTS: Here, we exposed wild-type Drosophila melanogaster to BPA during embryonic and larval development. We used immunohistochemistry and confocal microscopy to examine axon outgrowth in the adult brain. We found that millimolar concentrations of BPA cause midline crossing defects in fasciculating axons of the mushroom body, an adult neural structure required for higher sensory integration. FUTURE DIRECTIONS: We are currently investigating how BPA exposure affects axon outgrowth in fmr1 mutant Drosophila to elucidate the gene x environment interaction of BPA and fmr1. We hypothesize that the BPA-mediated axon outgrowth phenotype will be significantly more severe in fmr1 versus wild-type Drosophila. FUNDING: CSU Sacramento Research and Creative Activities Award and Goethe Research Award.
Poster #: 125
Campus: Stanislaus State University
Poster Category: Product-focused Innovation
Keywords: Brewery yeasts, Cell fusion, CRISPR/Cas9
Project Title: Disruption of Mating-Type Switching in Brewing Saccharomyces cerevisiae Strains by CRISPR/Cas9 for Cell Fusion
Author List:
Kang, Shelly; Undergraduate, Biological Sciences, California State University, Stanislaus, Presenting Author
Kang, Brenda; Undergraduate, Biological Sciences, California State University, Stanislaus, Presenting Author
Kang, Ellerum; Undergraduate, Biological Sciences, California State University, Stanislaus
Kang, Choong-Min; Faculty, Biological Sciences, California State University, Stanislaus
Abstract: Since the Neolithic age, brewer’s yeast (Saccharomyces cerevisiae) has been widely utilized for food and beverage production. Pure cultures of S. cerevisiae have been utilized in brewing industry but current trends in brewing industry demands more variety of yeast strains that can be used to create new types of alcohol products.
The long-term goal of this project is to create hybrid S. cerevisiae strains of various industrial strains by cell fusion that have improved characteristics such as desirable flavors and increased ethanol production. To achieve this goal, we have collected fourteen industrial strains of S. cerevisiae including beer, sake, wine, and cider yeasts. All strains were tested for their sporulation in the SPO++ sporulation medium and seven strains successfully produced a- and α-haploid mating-types. Opposite mating-type haploids of some of these strains did not show mating-type switching, and are now being used to create hybrid strains. However, one strain, English Cider, sporulated to a- and α-haploids but switched their mating type and thus cannot be used for fusion. For example, pure a-haploid culture became diploid because some a-haploid offspring switched to α-haploid while replicating in the population. Thus, our short-term goal is to mutate mating-type switching gene (HO) in the English Cider strain by CRISPR/Cas9 system. Once ho mutants of the strain are successfully made, they will be used to sporulate and fuse with other haploid strains.
More than fifteen mutant alleles of ho have been known. However, the most common mutations among the ho mutants were found at nucleotide position of 1424, 1635, 1710 and 1740 of the standard lab strain of S. cerevisiae. DNA for guide RNA (gRNA) targeting the PAM (protospacer adjacent motif) sequence of each nucleotide position was created and cloned into a pCAS plasmid. Double-stranded DNA repair oligonucleotides were also generated by PCR. The pCAS plasmid containing gRNA and linear repair DNA were co-transformed into competent cell of the English Cider strain. Through this approach, we have successfully made a mutation at 1635. We are currently working on other ho mutation sites (1424, 1710 and 1740). Once all mutations are obtained we will test their sporulation and mating-type switching. Those haploids of the mutants that do not switch mating-types then will be used to fuse with the opposing mating-type of another brewing strains to make hybrid strains.
Poster #: 126
Campus: Sonoma State University
Poster Category: Product-focused Innovation
Keywords: rf power harvesting, battery-less sensor, implantable sensor
Project Title: Radio-Frequency Power Harvester for Sensors
Author List:
Russell, Brandon; Undergraduate, Engineering Science, Sonoma State University, Presenting Author
Normantas, James; Undergraduate, Engineering Science, Sonoma State University, Presenting Author
Gonsalves, John; Undergraduate, Engineering Science, Sonoma State University
Abstract: Cell phone signals are in the air around us. This small but persistent ambient radio-frequency (RF) power is modulated with data, yet in our Dean’s Summer research at Sonoma State University (SSU), we aimed to harvest this omni-present surplus of power irrespective of the data transferred. This largely unharvested ambient power can be particularly useful to address the CHALLENGE of energizing wearable and implantable sensors, in addition to energizing small sensor nodes in environments where batteries may present a hazard.
The RF power harvester (RFPH) was designed to capture ambient RF power and convert it to DC voltage stored in a super capacitor. The GOAL of the designed and built RFPH is collecting power from GSM signals at 830.4 MHz. The RFPH is comprised of three building blocks: 1) Impedance tuned receiver antenna, 2) 4-stage rectifier/charge pump with super capacitor, and 3) a low-power LED to test the operation. The METHOD of realizing the RFPH started with circuit simulation of the rectifier/charge pump in ADS software, and a rectangular patch antenna in FEKO software. Field measurement of ambient RF power in the hallways of the department showed availability of up to -17.73 dBm of ambient power. The rectifier/charge pump is matched to the antenna via a stub network and fed into 35F storage capacitor. Simulation results showed a working efficiency of 40%. Measurement RESULTS using a FieldFox Spectrum Analyzer showed an efficiency of 35.7% with a 50.1 KΩ load. The realized results are promising, yet further investigation is needed to deploy the RFPH for implantable devices. Our NEXT STEPS include investigating the possibility of miniaturizing the device by changing the type of antenna used and using a storage capacitor of lower capacitance to save on device volume.
As an ADDED VALUE, during the course of this research, our team established a printed circuit board (PCB) etching lab at SSU. This lab allowed for rapid development of designs and revisions of the antenna and charge pump PCBs. As a result other SSU students were exposed to streamline methods for making their own PCB designs a reality. We share these developed etching methods in our research for PCBs trace etchings.
Poster #: 127
Campus: CSU San Marcos
Poster Category: Programmatic (Core, Stem Cell, Bridges, PSM)
Keywords: stem cells, reprogramming,
Project Title: NOVEL METHODS FOR RAPID ESTABLISHMENT AND SCALE UP OF IPSC
Author List:
Briggs, Amy; Undergraduate, Biology, California State University San Marcos, Presenting Author
Lakshmipathy, Uma; Thermo Fisher Scientific
MacArthur, Chad; Thermo Fisher Scientific
Dargitz, Carl; Thermo Fisher Scientific
Abstract: The development of reprogramming technology for the generation of induced pluripotent stem cells (iPSCs) has catalyzed powerful possibilities in the field of stem cells and regenerative medicine. For these applications, one of the enabling methods that is critical is the ability to scale up while reducing variability between users. Initial steps in the iPSC workflow typically requiring clonal selection and manipulation can be further streamlined to develop a robust process for iPSC establishment. This study explores the use of novel solutions for clonal versus bulk iPSC generation. Clonal isolation of single colonies is the traditional method for establishment of iPSCs. Sendai virus based reprogramming has shown to be consistent and robust thus allowing for pooling of the clones. iPSCs established through bulk expansion should therefore be comparable to clonally expanded iPSCs. A critical factor for a pooled iPSC generation and scale up was the need for mild cell harvesting methods. We identified a novel solution that facilitates gentle passaging across a broader window of manipulation. To confirm the comparability between iPSCs generated using clonal and bulk methods, the cells were monitored for growth rates and morphology. The resulting iPSCs were subjected to rigorous characterization using transcriptome analysis, in vitro differentiation potential, and genomic stability. Further, iPSCs generated using the two different methods were expanded in large scale cell culture system such as Nunc Cell Factories. The combination of these workflow improvements, with tools that allow for elimination of partially reprogrammed and Sendai virus-containing cells provides a streamlined scale up protocol that is also consistent.
Poster #: 128
Campus: San Francisco State University
Poster Category: Proteins (Include Proteomics)
Keywords: polyamines, acetyltransferases, chimeric proteins
Project Title: Construction and investigation of C-terminal and chimeric Vibrio cholerae SpeG proteins
Author List:
Le, Van; Graduate, Chemistry and Biochemistry, San Francisco State University, Presenting Author
Lim, Ee Qing; Undergraduate, Chemistry and Biochemistry, San Francisco State University
Lim, Ee Qi; Undergraduate, Chemistry and Biochemistry, San Francisco State University
Gawat, Darwin; Undergraduate, Chemistry and Biochemistry, San Francisco State University
Solis, Allan; Undergraduate, Chemistry and Biochemistry, San Francisco State University
Boeck, Paloma; Undergraduate, Chemistry and Biochemistry, San Francisco State University
Renolo, Rossellini; Undergraduate, Chemistry and Biochemistry, San Francisco State University
Ma, Huixin; ACS Seed Program (high school student)
Abstract: The polyamines spermine (spm) and spermidine (spd) are important polycationic molecules for many life processes including cell growth, protein synthesis and metabolic regulation. These molecules are acetylated by polyamine acetyltransferases, but the roles of these acetyltransferases across all domains of life are unclear. The most well-studied polyamine acetyltransferase is the human spermine/spermidine N-acetyltransferase (hSSAT), which adopts a different oligomeric state than bacterial SpeG polyamine N-acetyltransferases. In addition, SpeG is an allosteric protein, whereas to our knowledge hSSAT does not exhibit this characteristic. To elucidate the structural features that are critical for adopting a particular oligomeric state or allosteric behavior, we created twenty mutants, truncations, and chimerics between SpeG from Vibrio cholerae (VcSpeG) and hSSAT enzymes and measured their kinetic activity. We also analyzed their oligomeric states using Native PAGE. We found many of these constructs in the presence of the polyhistidine tag were unstable in absence of glycerol, several caused the enzyme to lose significant activity, selected point mutations retained similar activity to the wild-type protein, and some constructs caused a change in oligomeric state. Our next step is to cleave the polyhistidine tag from these constructs and re-evaluate the kinetics and oligomeric states to determine if presence of the tag alters these behaviors. Overall, our results provide a framework for assessing key elements that are required for bacterial polyamine acetyltransferase allosteric and oligomeric behavior.
Funding for this work was provided by a CSUPERB New Investigator Grant, a Presidential Award for Professional Development of Probationary Faculty, Startup funds from San Francisco State University, and the ACS 2018 Seed Program.
Poster #: 129
Campus: CSU Sacramento
Poster Category: Proteins (Include Proteomics)
Keywords: apolipoprotein, amyloid, protein folding
Project Title: Differential effect of acidic pH on stability and amyloid formation in wild-type and G26R apolipoprotein A-I
Author List:
Tran, Thao; Graduate, Chemistry, California State University, Sacramento, Presenting Author
Roberts, Linda; Faculty, Chemistry, California State University, Sacramento, Presenting Author
Abstract: Apolipoprotein A-I (ApoA-I) is the main protein of HDL, which transports cholesterol from peripheral tissues and has cardioprotective properties such as the ability to act as an anti-oxidant. Despite its beneficial features, certain mutations or solution conditions such as acidic pH increase the propensity for misfolding, leading to formation of insoluble deleterious fibrils that deposit in tissues and likely contribute to heart disease. A key factor in amyloid formation is stability of the protein. The influence of acidic conditions on protein stability was analyzed by incubating wild-type (WT) and amyloidogenic G26R ApoA-I for a period of 21 days at neutral and acidic pH using equilibrium solvent denaturation in urea. Protein aggregation was determined by light scattering and amyloid formation by ThT binding. The unfolding of the protein was monitored using intrinsic tryptophan fluorescence. Data were fitted to obtain free energy of folding, m (cooperativity), and D1/2 (midpoint of denaturation). The free energy at neutral pH is similar for WT and G26R apoA-I, with delta G values of 4.41 kcal/mol and 4.35 kcal/mol respectively. The D1/2 for G26R is higher than WT, 2.23 vs 2.7 M, and is accompanied by lower cooperativity. Exposure to acidic pH (5.5 and 6.0) caused immediate aggregation of both WT and G26R apoA-I, with greater aggregation occurring in WT protein. Aggregation of both proteins increased over time at acidic pH. The free energy substantially increased in WT apoA-I at pH 5.5 and 6.0 to 9.83 and 8.04 kcal/mol, respectively. The D1/2 and m values for WT apoA-I also increased. Incubation of WT protein at 37 degrees Celsius up to 21 days led to a decrease in free energy to about 7 kcal/mol; the D1/2 and cooperativity also decreased over time, indicating a change in aggregated structure. The increased stability of WT apoA-I at acidic pH is consistent with formation of amyloid structure which was confirmed by an increase in ThT binding of more than 10 fold from day 0 to day 21. In contrast, the free energy of unfolding for G26R at pH 6.0 was unchanged at day 0 and remained relatively constant over time. A much smaller increase in ThT binding in G26R was observed as was a lower intensity of light scattering compared to WT protein. These results indicate that WT apoA-I is more prone to amyloid formation at acidic pH and may explain why WT apoA-I deposits in its full-length form in the acidic microenvironment of atherosclerotic plaque.
Poster #: 130
Campus: CSU San Marcos
Poster Category: Proteins (Include Proteomics)
Keywords: protein-protein interaction, fluorescence spectroscopy, Curli
Project Title: Investigating the Oligomeric State of CsgE
Author List:
Kaur, Kuldeep; Undergraduate, Chemistry and Biochemistry, California State University San Marcos, Presenting Author
Rabi, Huda; Undergraduate, Chemistry and Biochemistry, California State University San Marcos, Presenting Author
Jayasinghe, Sajith; Faculty, Chemistry and Biochemistry, California State University San Marcos
Abstract: Introduction
Gram-negative bacteria, such as E.coli and Salmonella, contain proteinaceous, hair-like, cell surface organelles known as curli. Curli serve to facilitate cell-cell interactions and are essential for host cell colonization. Curli assembly involves six proteins, CsgA, CsgB, CsgC, CsgE, CsgF, and CsgG. It is thought that CsgE acts as chaperones to prevent the premature aggregation of CsgA and/or CsgB which are the main protein components of Curli. CsgE has been shown to form an oligomeric structure and we sought to determine the nature of this oligomerization using fluorescence quenching of labeled CsgE.
Methods
The BL21 bacterial expression system was used to express four different mutants of CsgE (34C, 58C, 99C and 119C) where the wild type residue at the respective position was replaced by a cysteine. Cell pellets recovered from protein expression were lysed using a French Press and then were purified using affinity chromatography. The cysteines were labeled using the environment sensitive probe IAEDANS, and purified again through affinity and gel filtration chromatography. Fluorescence quenching was used to determine the residues that may be involved in forming the oligomeric structure of CagE.
Results and Discussion
We measured quenching of IAEDANS by Potassium Iodide (KI) at various CsgE concentrations. At concentrations above 2 uM we observe a Stern-Volmer quenching constant of less than 3 for IAEDANS labeled at position 119, while at concentrations below 2 uM the quenching constant increased significantly to greater than 7. The protection of the label from the quencher at higher protein concentrations could be due to the C-terminus of the protein involved in mediating protein-protein aggregation.
Poster #: 131
Campus: CSU Northridge
Poster Category: Proteins (Include Proteomics)
Keywords: chaperone, structure and function, NMR
Project Title: Investigation of HdeA mutants to better understand the mechanism of acid chaperone activation
Author List:
Geddes-Buehre, Dane; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Crowhurst, Karin; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract: The periplasmic protein HdeA is a known acid-stress chaperone, promoting survival of several species of bacteria in the low pH of the stomach, in turn allowing colonization of the intestines by the invasive bacteria. The colonization of bacteria in the intestines is detrimental to health, usually resulting in dysentery, which is frequently fatal in third world countries. Understanding the mechanism by which HdeA stabilizes other proteins in the periplasm is essential to future development of therapeutics to combat the potentially life-threatening activities of the chaperone.
HdeA is a pH sensitive chaperone that becomes activated only at low pH, preventing aggregation of the other periplasmic proteins that unfold in the acidic environment of the stomach. Previous work from our lab as well as others have shown HdeA becomes active near pH 2.4, dissociating from an inactive homodimer into active monomers. My current research project focuses on mutating key charged residues and identifying the resulting structural and/or functional changes at different pHs using primarily Nuclear Magnetic Resonance (NMR) spectroscopy. The mutation of glutamate 37 to valine resulted in alteration of numerous peak positions in 1H-15N spectra corresponding to residues far beyond the area surrounding position 37, and implying that the single mutation has a widespread impact on the tertiary structure of the protein. The deletion of five residues from the long flexible loop between helices B and C, a location hypothesized to be the site of initiation of dimer dissociation, caused the protein to unfold at a higher pH, and provided evidence for large perturbations in the folded dimer. The observed chemical shift differences may be the result of alterations in tertiary structure or they may reflect extensive changes in protein flexibility, which has functional implications. Further studies of structure and dynamics are necessary.
In addition to NMR analysis, the mutants were analyzed via pH titration using far-UV Circular Dichroism (CD). The secondary structure of the protein detected by CD confirmed the mutants unfolded at different pH values compared to the wild type, further highlighting the loss of stability that came from mutating key residues.
We gratefully acknowledge the support of the NIH for research funds (SC3-GM116745), as well as the NSF for funding the purchase of our NMR spectrometer (CHE-1040134).
Poster #: 132
Campus: CSU Northridge
Poster Category: Proteins (Include Proteomics)
Keywords: chaperone, protein-protein interaction, NMR
Project Title: Use of NMR techniques to describe the interactions of the chaperone protein HdeA with its substrate HisJ
Author List:
Benson, Jonathon; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Crowhurst, Karin; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract: HdeA is one of several proteins, found in the periplasm of acid tolerant bacteria such as E. coli, that confer acid resistance. Under physiological conditions HdeA is an inactive folded dimer. At the low pH of the human stomach, HdeA becomes an unfolded monomer and acts as a chaperone by complexing with acid sensitive proteins, such as HisJ, to prevent their aggregation. This chaperone action is ultimately key for the survival of pathogenic bacteria in the human gut. Understanding this mechanism is essential for eradicating diseases such as dysentery which are responsible for thousands of deaths annually.
The goal of our group is to understand and characterize the mechanism of activation, unfolding, and substrate binding of HdeA using modern nuclear magnetic resonance (NMR) techniques. Previous experiments determined that HdeA unfolds and is activated between pH 3.0 and 2.0. My project’s current goal is to describe the interactions between HdeA and a client protein, HisJ, as the proteins transition from physiological pH to the low pH conditions at which HdeA is activated. Our hypothesis is that, by titrating increasing quantities of substrate while monitoring resulting structural and dynamic changes with NMR, we can obtain an atomic level chaperone-substrate model for HdeA. To that end, protein-protein interactions were monitored at amide positions using 1H-15N correlation NMR experiments with increasing ratios of HdeA:HisJ at incremental pH values from pH 3.0 to pH 2.0. Initially, at pH 3.0, peak intensities of glutamine side chain amides increased significantly with the introduction of HisJ. At pH 2.4 (where HdeA is known to be activated), a greater number of residues showed increased peak intensities, suggesting interaction locations with HisJ. Of interest was the increase in the BC loop region of HdeA, which is also proposed to be the site of initiation of chaperone activation. These results indicate that early client interaction with exposed glutamine side chains may be the first chaperone-substrate interplay. Additionally, evidence for changes in the BC loop region at pH 2.4 may suggest substrate involvement in the HdeA activation process. Further research will continue to explore changes in peak intensities and protein motions at varying pH, as HisJ is titrated into solution.
We gratefully acknowledge the support of the NIH for research funds (SC3-GM116745), as well as the NSF for funding the purchase of our NMR spectrometer (CHE-1040134).
Poster #: 133
Campus: CSU Northridge
Poster Category: Proteins (Include Proteomics)
Keywords: chaperone, acid stress, NMR
Project Title: Use of NMR to probe changes in the flexibility of acid-stress chaperone HdeB from its inactive to active state
Author List:
Abasi, Lannah; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Crowhurst, Karin; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract: HdeB is an intrinsically disordered periplasmic chaperone essential to the acid resistance of numerous pathogenic bacteria. It, along with its homolog, HdeA, binds to vulnerable periplasmic proteins at low pH and prevents them from irreversibly aggregating in the acidic stomach. The activity of these two proteins is responsible for the survivability of pathogenic bacteria and resulting proliferation of dysentery. At physiological pH, HdeB is an inactive folded homodimer and remains a structurally unchanged, fully folded dimer in its active state (at pH 4-5), suggesting that the mechanism of chaperone activation is a result of changes in its dynamics (internal motions).
The long-term goal of this study is to gain insight into HdeB’s unique chaperoning mechanism by characterizing its pH dependent changes in structure and dynamics. In order to achieve atomic level specificity and detect subtle conformational changes, we are utilizing Nuclear Magnetic Resonance (NMR) spectroscopy to compare HdeB’s flexibility and structure in its inactive and active states at pH 6.0 and 4.5, respectively. Isotopically labeled HdeB was recombinantly expressed and purified. Subsequently, protein backbone and side chain dynamics were examined at pH 6 and pH 4.5 utilizing R2 and R1 relaxation experiments, which identify fast (ps–ns) time scale motions within the structure; this indicates local motions, such as methyl rotations. Additionally, intermediate (µs-ms) time scale motions, which reflect global conformational changes and flexibility, were identified using CPMG relaxation dispersion experiments. Notable changes in dynamics were observed with activation. At both pHs, intermediate time scale dynamics are clustered at the dimer binding interface (BC loop and helix B) as well as portions of helix C adjacent to helix B. With the transition to pH 4.5, the I55 side chain and the backbone at T40, T33, T22, G37 and K53 also gain µs-ms timescale motions. This suggests an increased surface area of flexibility around the dimer binding interface, which may enable interactions crucial to substrate binding. Continuing research will focus on determining whether solvent accessibility is altered during activation via hydrogen deuterium exchange experiments.
We gratefully acknowledge the support of the NIH for research funds (SC3-GM116745) and BUILD PODER for research support to L.A., as well as the NSF for funding the purchase of our NMR spectrometer (CHE-1040134).
Poster #: 134
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Lewis base, organocatalysis, C-H functionalization
Project Title: Investigative Mechanistic Studies of Lewis Base Catalyst-Controlled Regioselective Chlorination of Arenes and Heterocycles
Author List:
Dinh, Andrew; Graduate, Chemistry & Biochemistry, San Diego State University, Presenting Author
Janke, Laleña; Undergraduate, Chemistry & Biochemistry, San Diego State University, Presenting Author
Brown, Zach; Graduate, Chemistry & Biochemistry, San Diego State University
Maddox, Sean; Postdoc, Chemistry & Biochemistry, San Diego State University
Addison, Bennett; Faculty, Chemistry & Biochemistry, San Diego State University
Gustafson, Jeffrey; Faculty, Chemistry & Biochemistry, San Diego State University
Abstract: Aryl chlorides are numerous in drug discovery as synthetic handles for late-stage functionalization. Electrophilic aromatic substitution (EAS) is a common method of synthesizing such aryl chlorides. However, this method for C-H functionalization is hindered by harsh reaction conditions, reduced reactivity towards electron poor substrates and an inability to control the regioselectivity of the products due to the innate electronic properties of various substrates. Previous work by Miller has produced regio-specific halogenation on natural products and small molecules through a catalytic peptide, while Lewis developed a bio-catalytic enzyme mutation that overcomes regioselectivity. Despite such progress, it would be synthetically useful, especially in the field of medicinal and process chemistry, to have access to a mild catalytic-controlled regioselective halogenation methodology.
Herein, we have developed a Lewis base catalyst-controlled regioselective method for the C-H chlorination of simple phenols. Numerous sulfur and selenide-based catalysts (i.e. triphenylphosphine sulfide, Nagasawa’s thiourea) were tested, providing high ortho and para selectivity of up to 27:1 and 20:1, respectively. While investigating the mechanism of action, we observed the formation of catalytic intermediates, such as a triphenylphosphine dimer and a guanidine derived from Nagasawa’s catalyst. We hypothesize that these intermediates could possess catalytic activity to primarily drive product formation. To investigate the true catalytic species, we performed H1 and P31 kinetic experiments via nuclear magnetic resonance (NMR) spectroscopy to measure the initial rate of reaction across a variety of substrates comparing all four proposed catalytic species. Across all substrates, the triphenylphosphine monomer catalyst was 4-5 times faster than the dimer, with the fastest being 15 times faster, indicating that the dimer is not the primary catalytic species and its formation during the reaction is likely a factor in slowing down the rate. Similarly, a slower rate of reaction for the guanidine intermediate also indicates that our initial hypothesis was incorrect; further kinetic studies are needed for verification. Future studies include the isolation of crystal structures, as well as utilization of computational calculations and models such as Gaussian to determine relative energy values of transition state for the intermediates.
Poster #: 135
Campus: CSU Fresno
Poster Category: Synthetic Chemistry
Keywords: zampanolide, anticancer agent, Natural Product
Project Title: Synthesis of an Advanced Intermediate towards Macrocyclic Ketone Mimic of Zampanolide
Author List:
Jiang, Ziran; Graduate, Chemistry, California State University, Fresno, Presenting Author, Eden Award Nominee
Davis, Lauren; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Chen, Guanglin; Staff, Chemistry, California State University, Fresno
Chen, Qiao-Hong; Faculty, Chemistry, California State University, Fresno
Abstract: (-)-Zampanolide is a naturally occurring macrolide that was isolated from two species of marine sponge. It has been established as a very attractive anticancer lead compound due to its unique covalent-binding with tubulin and low nanomolar antiproliferative potency even against multi-drug resistant cancer cell lines. In addition to the limited supply, we envision that the lactone moiety is not drug-like enough due to the metabolically instability. This study thus aims to synthesize a stabilized and simplified zampanolide mimic by replacing the lactone core structure in zampanolide with a macrocyclic ketone, as well as by removing the tetrahydropyran ring.
To this end, a highly advanced intermediate towards the zampanolide mimic has been successfully achieved through a Horner-Wadsworth-Emmons reaction of Fragment C13-C18 branched with C17-CH2-C1-C2 and Fragment C3-C8. The Fragment C3-C8 was built up from commercially available 2-butyn-1-ol via a seven-step transformation. Fragment C13-C18 branched with C17-CH2-C1-C2, with an appropriate functions group (beta ketone phosphate) installed, has been successfully constructed via a twelve-step sequence. Most importantly, the critical C17-CH2-C1=C2 bond for the macrocyclic ketone mimic of zampanolide has been constructed by a cross-coupling akylation mediated by CuI/TMEDA/LiOMe. The double bond on C1 and C2 have been converted to a beta ketone phosphate using Miyaura borylation as the critical reaction. All synthesized intermediates have been characterized by interpreting the 1H and 13C NMR spectra. This project is supported by NIH/NIGMS under Award Number SC2GM121185.
Poster #: 136
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Drug Synthesis , atropselectivity, photocatalysis
Project Title: Rational Drug Synthesis via Asymmetric Suzuki Reaction applying photocatalytic Perovskite and Nickel(II) Complex
Author List:
Zhu, Xiaolin; Postdoc, Chemistry and Biochemistry, San Diego State University, Presenting Author
Yan, Yong; Faculty, Chemistry and Biochemistry, San Diego State University, Presenting Author
Zhu, Dian; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Sun, Yue; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Abstract: Cost-effective and efficient photocatalysis are highly desirable for the synthesis of drugs, biomolecules, advanced materials etc. Based on the remarkable advances in photocatalysis, the development of photocatalytic protocols has been made for artificial C-C, C-O and C-N bond formations. However, many current photocatalysts require: precious metals (i.e. Ir, Ru, etc); complicated synthetic preparations or demonstrate moderate activity and are thus, not desirable. The outstanding performance of lead-halide perovskites in photoelectrons can be attribute to the high absorption coefficient, high fluorescence quantum efficiency and long excited state lifetimes, which are also required for efficient photocatalysis. We reported that perovskites nanocrystals (NCs) of APbX3 (A = Cs, MA, X = Cl, Br, I) are very promising photocatalysts that can effectively photocatalyzed a-alkylation of aldehydes under visible light.
Suzuki-Miyaura cross-coupling is one of the most important C-C formation reactions, which rely on expensive palladium complexes. In this work, we explore a dual catalysis system that the merger of readily synthetic perovskite APbX3 nanocrystals with economical Ni(II) complex to formation Csp2-Csp2 bonds by the cross-coupling of aryl halides with arylboronic acids. The reaction has been optimized using methyl 4-bromobenzoate and phenylboronic acid as starting materials, in the presence of 1 mg APbX3, 10mol % of Ni(II) salt and 2 equiv. of base at room temperature, under the irradiation with Blue LED to afford 86% of desired product. Room temperature of Suzuki reaction renders further asymmetric synthesis of drug molecules via atropselectivity. Furthermore, an anticancer drug lapatinib has been synthesized in three steps with a 55% overall yield, indicating a highly practical synthetic methodology. Photophysical studies revealed that the excited-state nickel(II) produced through energy transfer from the excited photosensitizer perovskite NCs are responsible for the facility of reductive elimination. Comparing to the rare, high-price noble transition-metals, our cost-effective, easily-prepared, highly-active and band-tunable hybrid halide perovskites are alternative choices in the photocatalysis of organic reactions.
Funding acknowledgements: NSF under Chemical Catalysis program, award 1764142; San Diego State of University start-up funding.
Poster #: 137
Campus: San José State University
Poster Category: Synthetic Chemistry
Keywords: Enzyme mimics, Polymers, Catalysis
Project Title: Mimicking secondary sphere interactions around biological enzyme active sites using single chain polymeric nanoparticles
Author List:
Gupta, Vanshika; Undergraduate, Chemistry, San José State University, Presenting Author
Nguyen, Khanh; Undergraduate, Chemical and Materials Engineering, San José State University, Presenting Author
Chua, Charleston; Graduate, Chemical and Materials Engineering, San José State University
Abstract: Enzymes, molecules that act as nature’s catalysts, facilitate the vital reactions of biology, which are often also relevant to the production of commercial chemical products. The complexity of many enzymes, however, precludes their synthesis on an industrial scale. Non-biological small molecule catalysts can be designed to mimic an enzyme’s active site, but without the proteinaceous structure, these complexes often fail to achieve enzyme-like reactivity. Thus, our work intends to mimic the protein structure around an active site through the synthesis of a single chain nanoparticle (SCNP) made from an ABA triblock polymer. Block polymer describes a macromolecule wherein a series of one monomer is followed by a series of a different monomer and can combine the properties of both in a single material. For our synthesis, we began by making homopolymers with each monomer via reversible addition-fragmentation chain-transfer polymerization. After purification, we characterized our poly(2-hydroxyethyl acrylate) (PHEA), poly(tert-butyl acrylate) (PtBA), and poly(N-isopropylacrylamide) (PNIPAM) with size exclusion chromatography (SEC) and nuclear magnetic resonance spectroscopy (NMR), confirming that we had successfully made each material and quantifying the average size of the polymer chains. In the SCNP, PHEA will serve as the hydrophilic A block, while a copolymer of tBA and NIPAM will serve as the hydrophobic B block. The NIPAM units will provide hydrogen bond donors to accommodate and interact with substrates that react at the catalytic center of our SCNP. To complete our SCNP, we will covalently attach a transition metal complex. Poly(tBA-co-NIPAM) was successfully synthesized with a 4:1 ratio of the two monomers, based on NMR analysis. Block polymers with PHEA and PNIPAM or PtBA and PNIPAM have been produced through sequential polymerization steps and analyzed by NMR and SEC. The final block polymer targets are block polymers of PHEA with PtBA or (PtBA-co-NIPAM), which have proven challenging to make and to characterize based on the strong incompatibility between PHEA and PtBA. Now we are modifying the polymers such that a terminal thiol group will be available to connect via thiol-ene click chemistry to an alkenylated metal complex. By mimicking a biological enzyme with SCNPs we aim to enable enzyme-like catalysis and to gain insight into secondary sphere interactions of natural enzymes. We acknowledge San José State and CSUPERB for funding.
Poster #: 138
Campus: CSU Fresno
Poster Category: Synthetic Chemistry
Keywords: Anticancer agent, zampanolide, synthetic medicinal chemistry
Project Title: Synthesis towards Lactam Mimics of Zampanolide
Author List:
Gonzalez, Maricarmen; Graduate, Chemistry, California State University, Fresno, Presenting Author
Chen, Guanglin; Staff, Chemistry, California State University, Fresno
Chen, Qiao-Hong; Faculty, Chemistry, California State University, Fresno
Abstract: Zampanolide is a marine macrolide that exhibits low nanomolar cytotoxicity against both drug-sensitive and multi-drug resistant cancer cell lines. This new member of microtubule stabilizing agents (MSAs) causes microtubule polymerization which in turn leads to cell apoptosis in the G2/mitotic phase of the cell cycle. Zampanolide’s microtubule stabilization is promoted through its unique covalent binding to β-tubulin which is found in microtubules. However, the metabolic instability of zampanolide is attributable to the lactone bond cleavage catalyzed by esterase. Therefore, our goal for this project is to hopefully improve zampanolide’s metabolic stability through the manageable synthesis of simplified lactam mimics. At this point, fragment C1-C8 has been successfully synthesized in a total of 10 steps of reactions and fragment C13-C18 in seven steps, both starting with commercially available 2-butyn-1-ol. Furthermore, an amine group has been incorporated to fragment C13-C18 at the position of C17. This leads us to the position that is very close to the construction of the critical amide bond through the fusion of both fragments and our simplified lactam mimic core.
This project is supported by NIH/NIGMS under Award Number SC2GM121185.
Poster #: 139
Campus: San José State University
Poster Category: Synthetic Chemistry
Keywords: photoaffinity, Pseudomonas aeruginosa, synthesis
Project Title: Uncovering the Molecular Target of an Antipyocyanin Compound Using a Photoaffinity Labeling Approach
Author List:
Aboulhosn, Kareem; Undergraduate, Chemistry, San José State University, Presenting Author
Ortega, Dominic; Undergraduate, Chemistry, San José State University, Presenting Author
Moore, Rebecca; Graduate, Chemistry, San José State University
Cheng, Zi Jun; Undergraduate, Chemistry, San José State University
Miller Conrad, Laura; Faculty, Chemistry, San José State University
Abstract: Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that can cause life-threatening infections for those with weakened immune systems. Due to the rise in antibiotic resistance, new methods are needed to combat the bacterium. Our lab is interested in developing an antivirulence approach, where the production of virulence factors, the harmful products exported by a bacterium, is inhibited. The disarmed bacteria could then be cleared by the host’s immune system. P. aeruginosa produces a variety of virulence factors during an infection, including a redox-active small molecule called pyocyanin. We previously developed a small molecule that disrupts production of pyocyanin, however, the mechanism of action is unknown. In the current work, we aim to identify the molecular target of the pyocyanin inhibitor using a photoaffinity labeling (PAL) approach. Incorporation of a photoaffinity tag into a compound allows the formation of a covalent bond between the small molecule and its protein target. We desired an analog of the pyocyanin inhibitor that has a diazirine photoaffinity label and a terminal alkyne, to enable addition of a biotin label for affinity chromatography. We first synthesized analogs with the alkyne at several locations and tested each compound for antipyocyanin activity. We determined that a meta-substituted alkyne was the most successful, leading to only a small decrease in efficacy (95% vs. 100% reduction of pyocyanin at 100 µM). We then designed an analog that incorporates both a meta-substituted alkyne as well as a diazirine photoaffinity label. We will present our current synthetic progress toward our target PAL analog. We have successfully completed the first two steps in the synthesis. The third step involving a Grignard formation and subsequent iron-mediated acylation has been successful in a model system. Future plans to complete the synthesis will also be discussed. Upon completion of the PAL analog and labeling studies, labeled proteins will be identified by MS-MS to determine potential targets that will be confirmed in additional studies. We ultimately aim to characterize new processes that control pyocyanin production. This information could provide new targets for future antivirulence therapeutics to combat this life-threatening pathogen. The work was supported by the CSUPERB Presidents’ Commission Scholars Program (KA), NIH RISE (DO), LSAMP (DO), and NIH Grant 5SC3GM118199.
Poster #: 140
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Cyanobacteria metabolite, Anti-tumor, Solid-phase peptide synthesis
Project Title: Synthesis of the Correct Enantiomeric Form of Micromide, a Highly Potent Anti-Cancer Agent from Cyanobacteria
Author List:
Nelson-Hall, Tyler; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Molina, Josiah; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Wang, Lee; Staff, Chemistry and Biochemistry, San Diego State University
Bergdahl, Mike; Faculty, Chemistry and Biochemistry, San Diego State University
Hecht, David; Faculty, Chemistry and Biochemistry, San Diego State University
Abstract: Background: Over many years marine cyanobacteria have provided an array of biologically active products useful to combat various types of cancers. Among these is a secondary metabolite isolated from the benthic cyanobacteria Moorea producens, formerly classified as Lyngbya majuscula. Lipophilic extracts that showed activity against solid tumors were fractionated using bioassay-guided fractionation, spearheaded the discovery of micromide in 2004. Micromide displays an impressive potency against KB cells (IC50 = 260 nM). Micromide is a linear lipopeptide which consists of a modified thiazole head, a five residue peptide body and an aliphatic tail. Typically, lipo-peptide natural products have shown cytotoxicity through various mechanisms such as membrane disruption and microtubule inhibition, however, the mechanism of micromide has yet to be determined. Natural products from marine cyanobacteria provide the challenge of accessibility, as they are difficult to harvest and provide little material for scrutiny. In addition, these secondary metabolites vary from strain to strain, and may not be present in a harvested sample. Total synthesis also determines the accuracy of stereochemistry of proposed structures. Therefore, synthetic versions of micromide are critical for the elucidation and confirmation of the absolute stereochemistry of the isolated micromide harvested from cyanobacteria.
Results: Based on the structure originally published by Williams in 2004, we have synthesized micromide analogs using highly enantioselective methods to find that the NMR spectra did not match that of the proposed stereochemical structure of the natural product. Recently, the Tian group at the Hebei University independently synthesized micromide and published their findings. Both the spectra published by the Tian group and ours were identical. However, Tian erroneously concluded the synthetic product was micromide without further testing. The NMR spectrum did not match that of the natural product. In addition, our testing showed absolutely no biological activity against KB cells. We suspected that the synthetic product is an epimer of the natural product and we report the successful total synthesis of epi-micromide. Due to the lengthy nature of solution-phase chemistry, we have adapted our synthesis using solid phase peptide synthesis methodology. Microanalyses confirmed that the product is identical to the solution-phase product.
Poster #: 141
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: fluorescent probes, biophysics, nucleic acids
Project Title: A Solvatochromic Nucleoside to Report on Point Mutations and DNA Conformation
Author List:
Islas, Irazema; Undergraduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Liddle, Hannah; Undergraduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Wyllie, McKenzie; Undergraduate, Department of Chemistry and Biochemistry, San Diego State University
Abramowitz, Michael; Undergraduate, Department of Chemistry and Biochemistry, San Diego State University
Purse, Byron; Faculty, Department of Chemistry and Biochemistry, San Diego State University
Abstract: Nucleic acids play a key role in not only the storage of the genetic code, but they also regulate the code’s expression and play active roles in the metabolic chemistry occurring in cells. A variety of DNA sequence methods exist for studying nucleic acids, but there is still a great need for rapid, cheap, and robust methods to discriminate DNA sequences in complex mixtures. To that end, our lab is working on making a solvatochromic nucleoside analogue that has the potential to sense sequence with a color change. Building on our lab’s research on nucleoside analogues, we synthesized a tricyclic derivative of cytidine, dinitro-tCO, as a possible solvatochromic nucleoside. To synthesize dinitro-tCO we started with a commercially available 2′-deoxycytidine derivative and extended it into a tricyclic system using SNAr chemistry. After chromatographic purification, we confirmed the synthesis of this compound using 1H NMR, observing an anomeric peak at 6.23 ppm and the expected arene signals at 7.4–7.43 ppm. To test its solvatochromicity, we chose four solvents of varying polarity: 1,4-dioxane, acetone, isopropanol, and methanol. Our test resulted in methanol and 1,4-dioxane having the largest Stokes shift, indicating that the compound has strong solvatochromicity. Our next step is to insert the nucleoside analogue into DNA. The solvatochromic properties will allow to see changes in the conformations and folding patters of DNA and single nucleotide polymorphism. Laser wave mixing will then be used to maximize detection sensitivity. This method could be quick, inexpensive and have a high fidelity for DNA sequence.
Poster #: 142
Campus: CSU Northridge
Poster Category: Synthetic Chemistry
Keywords: chemotherapy, synthesis, membrane transport
Project Title: Anti-tumor activity of terpyridine complexes and polypyridine ligands targeting active transport processes
Author List:
Terzian, Armenuhe; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Melgar, Estelli; Undergraduate, Biology, California State University, Northridge, Presenting Author
Kelson, Eric; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract: Tpys (2,2’:6’,2”-Terpyridines) have been reported as promising anti-tumor agents that interfere with DNA replication and transcription by intercalation (inserting themselves into the base-pair stack). Interestingly, Tpys exhibit selectivity for tumor cells over normal ones suggesting exploitation of an active means of cellular uptake elevated in cancer. Our group has prepared Tpy-based ruthenium complexes bearing DNA intercalating groups as new anti-tumor candidates designed to exploit active transport. Specifically, four complexes have been investigated in detail: (Tpy)Ru(TpyTpy)(2+) (1) [where TpyTpy=4’,4””-bis(2,2’:6’,2”-terpyridine), (Tpy)Ru(Dppz)Cl(+) (2) (where Dppz= dipyrido[3,2-a:2′,3′-c]phenazine), (AnthTpy)Ru(Bpy)Cl(+) (3) [where AnthTpy=4’-(9-phenanthryl)-2,2’:6’,2”-terpyridine and Bpy=2,2’-bipyridine], and (AnthTpy)RuCl2(DMSO) (4) (where DMSO=dimethylsulfoxide). All four complexes bind DNA strongly (with the last three by intercalation) and exhibit significant toxicity (with values of EC50 from 30 to 6 µM) toward several tumor lines (MDA-MB-231, MDA-MB-468, MCF-7, and HepG2). In tests with active plasma membrane transport inhibitors, the toxicity of 1 was attenuated by added spermine suggesting assisted entry into tumor cells via polyamine transporters. This is consistent with the repetition of nitrogen donors within the pendent Tpy group chemically resembling natural polyamines. In contrast, the toxicities of 2, 3 and 4 (that bear no free Tpy groups) were unchanged by spermine but increased with chloroquine suggesting transport by endocytosis and release from endosomes with increased pH. These results inspired the design of new anti-tumor candidates bearing terpyridine-like and intercalating features. 2,3-Bis[(E)-(2-pyridyl)methylideneamino]phenazine (5) was prepared and found to bind DNA. Surprisingly, it exhibited little toxicity (EC50s greater than 100 µM) toward tumor cell lines (MDA-MB-231, MDA-MB-468, and MCF-7) in spite of having a terpyridine-like arrangement of nitrogens. The intense fluorescence of 5 indicates the molecule is inflexible and may not adopt conformations needed to bind polyamine transporters. To address this, the structurally similar but flexible 9,10-Bis{[(2-pyridyl)methyl]imino}-4,5-diazaphenanthrene (6) was prepared. Its DNA binding and toxicity will be presented. These efforts to target cancer through their elevated transport processes may lead to more effective chemotherapies with fewer side-effects.
Poster #: 143
Campus: CSU Long Beach
Poster Category: Synthetic Chemistry
Keywords: drug delivery, metal-organic framework, surface chemistry
Project Title: Drug encapsulation by surface supportive porous crystalline structures
Author List:
Bui, Angela; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Dinh, John; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Tian, Fangyuan; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract: Coronary artery disease contributes to 25% of all deaths nationally. To treat symptoms of this disease, polymer-based drug eluting stent coatings have been developed and have exhibited great advantages over bare metal stents alone. However, clinical studies have shown the polymer coating has been linked to numerous cases of hypersensitivity reactions and inflammatory responses. Our goal is to replace the polymer with an iron-based metal organic framework (Fe-MOF) due to its non-toxicity, high porosity, and biodegradable characteristics. Specifically, we used Material from Institute Lavoisier-88B (MIL-88B), which is composed of iron (III) trimers connected by aromatic dicarboxylate groups, forming a 3D cage-like structure. Our approach involves a three-step method starting with the synthesis of MIL-88B; followed by using stainless-steel (SS) as a metal substrate to obtain a uniform surface-supportive thin film using a direct crystallization method. Subsequently, we used ibuprofen as a model drug to test for its capabilities of drug encapsulation and release kinetics. X-ray photoelectron spectroscopy and Fourier Transform Infrared (FT-IR) Spectroscopy was applied to analyze the elemental composition, as well as, for chemical characterization of the thin film. The carboxylate groups from the FT-IR data indicates that MIL-88B is chemically bonded to the SS. Through a simple submersion technique, an ibuprofen/hexane solution was incubated for 24 hours. Additionally, FT-IR spectroscopy confirmed ibuprofen was loaded into the Fe-MOF through a series of C-H peaks ~3100 cm-1. We also observed that the Fe-MOF thin film on SS was composed of the elements: Fe, Cr, Co, C, and O. Using an optical tensiometer, the water contact angle was measure, and a tunable wettability of the Fe-MIL-88B film was observed after incubation of hydrophobic guest molecules. Currently, the drug loading capacity is being analyzed by UV-Vis spectroscopy and drug release kinetics by HPLC. In summary, a Fe-MOF thin film was grown onto SS and demonstrated successful encapsulation of ibuprofen into the MOF structure. Our preliminary findings are significant and show promise in increasing the understanding of MOF thin film formation on a metal substrate and its role in a drug delivery system.
Poster #: 144
Campus: CSU Fresno
Poster Category: Agriculture/Biofuels/Environment
Keywords: nematicide, C. elegans, chalcones
Project Title: Synergistic effects of linked chalcones on Caenorhabditis elegans
Author List:
Cortez , Chemyn; Undergraduate, Biology, California State University, Fresno, Presenting Author
Echeveria, Dustin; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Aujla, Gursharnjit; Undergraduate, Biology, California State University, Fresno
Gill, Jasmeet; Undergraduate, Biology, California State University, Fresno
Kim, Minjoo; Undergraduate, Chemistry and Biochemistry, California State University, Chico
Arpin, Carolynn C.; Faculty, Chemistry and Biochemistry, California State University, Chico
Calderón-Urrea, Alejandro; Faculty, Biology, California State University, Fresno
Abstract: Agriculture has been an essential part of human life since its development thousands of years ago. Despite its longtime existence, scientists are constantly advancing agricultural techniques and practices. One of the issues faced today is the presence of plant parasitic nematodes (PPNs), which account for billions of dollars in losses every year. Methyl bromide was used previously as a nematicide to control the subsistence of PPNs such as Meloidogyne incognita, but was deemed harmful to humans and the environment. Chalcones 17, 25, and 30 (characterized by the presence of an aromatic ketone and an enone at their core) were tested to analyze their effectiveness as alternative nematicides and were found to be 100% effective in killing nematodes at 10-4 M concentrations. Further experiments showed that, when these chalcones were mixed together in solution, a synergistic effect was observed, causing more worms to be killed at lower concentrations. The presented study aims to link two different chalcones in one molecule (17+25, 25+30, and 17+30) to test the idea that these linked chalcones can be as potent, if not more potent than, the individually mixed chalcones. We have thus developed a linear four-step synthesis that is modular and accommodating of various linkers, and we successfully prepared a small library of linked chalcones that is currently being assessed for their nematicidal activity. We are currently pursuing other libraries that differ in linker length and in position of linkage. Project motivation, synthesis design, troubleshooting, our library of final compounds, and biological data will be presented.
Poster #: 145
Campus: Sonoma State University
Poster Category: Agriculture/Biofuels/Environment
Keywords: bioenergy, wastewater treatment, microbial fuel cells
Project Title: A microbial fuel cell-based system for on-site treatment of winery wastewater
Author List:
Ramirez, Irving; Undergraduate, Biology, Sonoma State University, Presenting Author
Westover, Taylor; Undergraduate, Biology, Sonoma State University, Presenting Author
Hughes, Natalie; Santa Rosa Junior College
Kainuma, Mami; Okinawa Institute of Science and Technology
Simpson, David; Okinawa Institute of Science and Technology
Goryanin, Igor; Okinawa Institute of Science and Technology
Cohen, Michael; Faculty, Biology, Sonoma State University
Abstract: Owing to space limitations many small wineries are faced with the expensive alternatives of paying high sewage fees or trucking their wastewaters to off-site treatment facilities. Three years ago we established an experimental microbial fuel cell-based system for on-site treatment of winery wastewater (WW) at Vintners Square, Santa Rosa, CA. WW from a 900-L pH neutralization tank is pumped into a tubular MFC having a 45.3 L working volume from which it flows through a final polishing filter to irrigate landscaping. Conductive material inside the anaerobic anodic compartment of the MFC enables electron transfer between bacteria and methanogenic archaea, resulting in the production of biogas from organics in the WW, and creation of an external current that flows to an oxygen-reducing air-cathode. We monitored generation of biogas and electricity and changes in the concentration of organics in the WW, measured as chemical oxygen demand (COD). Operating for twelve months under a 5.7 day hydraulic retention time, the MFC removed 76.2 ± 12.2% (mean ± SD, n = 85) of the organics in the WW, corresponding to a removal rate of 0.50 ± 0.27 kg COD/m^3/d (mean ± SD, n = 19; equivalent to ~135 W/m^3). Production of biogas at 133 ± 97 L/m^3/d accounted for the majority of the removal activity (equivalent to ~0.38 kg COD/m^3/d). Only a small proportion of the energy from the WW went into formation of the external electrical current, generating 35.1 mW/m^3. Though small, the electrical output is a useful indicator for monitoring the microbial community health within the MFC. Consumption of acids across the MFC resulted in a rise in pH of 0.70 ± 0.28 (mean ± SD, n = 54) from inflow (mean pH 6.64) to effluent (mean pH 7.34). For four months at the end of the testing period the MFC effluent flowed through an earthworm-containing vermifilter that removed 35.2 ± 8.3% (mean ± SE, n = 6) of the residual organics from the effluent. Plant growth assays demonstrated that growth-inhibitory components still remain at the end of the treatment process. We are currently investigating means to target removal of these inhibitory compounds, isolating phenolic-degrading bacteria and fungi for use as potential inoculum of the vermifilter medium. Results gathered from the testing and optimization of this system will inform the development of full-scale MFC-based systems to treat agro-industrial wastewaters.
Poster #: 146
Campus: CSU Fresno
Poster Category: Agriculture/Biofuels/Environment
Keywords: Wastewater, Algae, biofuel
Project Title: Waste Water Quality Improvement by Algae Treatment
Author List:
Kang, Ravinder; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Adame, Isaac; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Maitra, Kalyani; Faculty, Chemistry, California State University, Fresno
Abstract: The nitrate and phosphate levels in ground water and aquifers have been on the rise due to the use of agricultural fertilizers, the seepage of wastewater from animal farm lagoons, and the use of animal waste as manure. High levels of these chemicals causes serious health issues, such as methemoglobinemia in infants and increases the risk of colon cancer in adults. Algal blooms can consume harmful nitrates and phosphates for growth; and wastewater from sumps at Fresno State farm have these two chemicals in high abundance. Chlorella sorokiniana and Chlorella vulgaris were grown in wastewater to show that the farm wastewater provides the required nutrients. The lipid content of these algal strains grown in these specific environment will be screened for their potential use in biofuel.
The collected wastewater was filtered through a 0.22-micron membrane filter to remove suspended particles and microbes. For one set of experiment, the water was circulated through a UV lamp (235.7nm) circulation system that was built in-house. For another set, the filtered water was directly used for culture inoculation.
The nitrate level in the water was analyzed at both pre- and post-algae growth stages. The pure algae cultures are initially grown in Bold 3N media in an aseptic environment under 24-hour aeration and LED light illumination (2100 lux). Measured amounts of these pure cultures were transferred to the bioreactor vessels, which contained 50/50 wastewater and media in one experiment and 100% wastewater in another. Both experiments were run at a controlled temperature of 25C, a pH level between 6 and 8, a total constant light intensity of 250 LED for 7 days, and growth was measured through optical density.
Pre-analysis of the water prior to inoculation showed nitrate levels of 4.3 mg/L for the filtered water and 2.4 mg/L for the UV exposed water. Post-analysis after algae harvesting showed a < 0.1 mg/L of nitrates in both sets. The data concluded that Chlorella vulgaris is successful in removing 95% of the nitrates in the wastewater. Another experiment with both the Chlorella species grown in UV exposed wastewater, under identical environment showed that Chlorella sorokiniana had the faster growth rate, reaching peak growth at 2 days. The lipid bodies for the algal strains from these experiments were qualitatively analyzed using BODIPY staining. It showed considerable lipid storage and therefore opens a scope to explore their potential use in biofuels.
Poster #: 147
Campus: CSU San Marcos
Poster Category: Agriculture/Biofuels/Environment
Keywords: chaparral, Nitrogen deposition, N-cycling bacteria
Project Title: Long-term nitrogen inputs cause a change in microbial abundance in semi-arid soils of Southern Californian
Author List:
Grant, Timothy; Graduate, Biological Sciences, California State University San Marcos, Presenting Author
Vourlitis, George; Faculty, Biological Sciences, California State University San Marcos
Abstract: Nitrogen (N) deposition alters the structure and function of terrestrial ecosystems, especially in urbanized Southern California where basins receive high N deposition from fossil fuel burning. Chronic N inputs are known to affect the soil carbon (C) and N cycling; however, the mechanisms are still poorly understood. Here we examined whether long-term experimental N deposition would alter the abundance of soil C and N cycling bacteria, and hypothesized that N inputs would cause a change in the abundance of key C and N cycling functional groups. Soil was collected from evergreen chaparral stand San Diego County, CA, and semi-deciduous coastal sage scrub (CSS) in Riverside County, CA, during the spring and fall of 2017. Both sites had paired-control and N-addition plots (n=4 plots/treatment) that had been treated with 50 kgN ha-1 y-1 since 2003. Microbial DNA was extracted from soil samples, purified, and sequenced for 16s RNA, and sequence data were identified to family and organized into C or N cycling functional groups. In chaparral, N inputs significantly increased the abundance of N2-fixing and denitrifying bacteria but reduced the abundance of nitrifying bacteria. In CSS, N addition significantly increased the abundance of denitrifying bacteria, but not nitrifying or N2-fixing bacteria, and also significantly increased the abundance of metal reducing bacteria. While nitrifying bacteria were either negatively impacted (chaparral) or unaffected (CSS) by added N, soil extractable NO3- increased by 2-10 fold in plots exposed to added N, suggesting that the increase in nitrification was due to an increase in microbial activity rather than abundance. Added N consistently increased the abundance of denitrifying bacteria, implying that long-term N addition stimulates gaseous N loss in these shrublands. These data have important implications for N storage in semi-arid shrublands exposed to long-term N deposition, and their contribution to atmospheric greenhouse gas concentrations.
Poster #: 148
Campus: CSU Fullerton
Poster Category: Agriculture/Biofuels/Environment
Keywords: Malus, genomics, transcriptome
Project Title: Enhancing reference genomes for cross-cultivar functional genomics in apples
Author List:
Cook, Brittany; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Honaas, Loren; United States Dept. Agriculture, Agricultural Research Service
Der, Joshua; Faculty, Biological Science, California State University, Fullerton
Abstract: Apples are one of the most valuable tree fruit crops in the United States, and modern advances in controlled-atmosphere storage has made apples commercially available year-round. However a number of economically important cultivars are susceptible to a number of storage disorders impacting post-harvest tree fruit quality. Understanding the physiology of these disorders has become an important component of apple breeding programs. While the apple genome was sequenced in 2010, the reference genome comes from a double haploid version of the Golden Delicious cultivar, but substantial genetic diversity among apple cultivars has represented a barrier to using genomic approaches in other economically relevant apple cultivars. We are using an RNA-seq and whole genome resequencing approach to characterize genetic diversity within apples and to improve the utility of the reference genome for cross-cultivar functional genomics. We have obtained and analyzed sequencing data from 62 samples of Honeycrisp (HC) and Granny Smith (GS) representing both healthy and diseased apples undergoing a number of storage treatments to identify genes related to post-harvest fruit quality. We selected 29 candidate genes, 15 from GS and 14 from HC, to explore in detail the genetic differences between cultivars of interest and the reference cultivar, Golden Delicious. Gene expression levels for these genes were also validated using qPCR. We hypothesis that correlation between genes of interest and hotspots of variation emphasizes the need to explore these differences in greater detail to move forward in applied, functional tree-fruit genomics. With a subset of transcriptome samples from GS, we evaluated the mapping rate of RNA-seq reads to the Golden Delicious reference. Reads were aligned to the reference using HISTAT2 and the transcript models were determined using StringTie. We found that only 70% of our apple transcriptome data confidently mapped to the reference genome, highlighting the need for an improved reference. We are building de novo transcript models using Trinity and have re-sequenced the GS genome using both Illumina and PacBio whole genome shotgun reads. We are in the process of assembling the GS genome and quantify the distribution of genetic variation between GS and Golden Delicious apples.
Poster #: 149
Campus: CSU Fresno
Poster Category: Agriculture/Biofuels/Environment
Keywords: Erigeron bonariensis, weed science, qPCR
Project Title: Characterizing the expression of candidate genes for herbicide resistance in the agricultural weed hairy fleabane (Erigeron bonariensis)
Author List:
Chaudhari, Priyanka; Graduate, Biology, California State University, Fresno, Presenting Author
Camarena Onofre, Diana; Undergraduate, Plant Science, California State University, Fresno, Presenting Author
Waselkov, Katherine; Faculty, Biology, California State University, Fresno
Abstract: Herbicide resistance is the heritable ability of weeds to survive and reproduce in the presence of herbicide doses that are lethal to the wild type of the species. Erigeron bonariensis (hairy fleabane) is an agricultural weed that infests orchards and crop fields in California’s Central Valley, and has become resistant to the herbicide chemical glyphosate (RoundUp®), through an unknown genetic mechanism. One mechanism of glyphosate resistance demonstrated in E. canadensis, a close relative of E. bonariensis, is non-target site reduced translocation of the herbicide, in which vacuolar sequestration prevents the chemical from spreading around the plant. This mechanism is believed to involve upregulation of the target gene EPSPS in combination with the ABC transporter genes M10 and M11. Our study aims to determine through quantitative PCR (qPCR) if these candidate genes are involved glyphosate resistance in wild populations of Erigeron bonariensis. Sample leaves of hairy fleabane were collected before and after glyphosate spraying in plants from 10 different populations wild-collected from the Central Valley and two control populations. Response to glyphosate was used to characterize percent resistance for each wild-collected population. RNA was extracted from the leaves of glyphosate-treated and untreated individuals, and used for cDNA synthesis. Quantitative PCR primers were designed for the E. bonariensis orthologues of the E. canadensis genes EPSPS, ABC M10, and ABC M11, and pre- and post-spraying expression levels of each gene (relative to the housekeeping gene actin) are currently being analyzed through qPCR. So far, results based on data collection from populations 1, 2, 5, and 9 suggest that in treated plants, there is high upregulation in M11 and EPSPS genes in plants sprayed at 1X or 2X field concentration of glyphosate, but unexpectedly, M10 is downregulated at 2X concentration. Completion of qPCR in Fall 2018 will establish whether the same candidate genes are involved in the glyphosate resistance response in E. bonariensis compared to its relative, and whether the variation in candidate gene expression among E. bonariensis populations correlates with variation in levels in resistance. Determination of the genetic basis of herbicide resistance will provide fundamental data about parallel evolution in response to strong selection pressures, and help suggest alternative mechanisms for field control of this weed.
Poster #: 150
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: Protein Design, Metal-Controlled Protein Complex Formation, Biophysical Charaterization of Designed Proteins
Project Title: Design of High-Affinity Metal-Controlled Protein Dimers
Author List:
Maniaci, Brian; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Huxford, Tom; Faculty, Chemistry and Biochemistry, San Diego State University
Love, John; Faculty, Chemistry and Biochemistry, San Diego State University
Abstract: The main goal of this research is to generate a metal-mediated protein interface and study the effects of interfacial modification on protein-protein affinity. Improving and enhancing protein interactions is of vital interest to the pharmaceutical and biotechnological industries. Incorporation of metal-binding sites at the target protein interface may be one approach to improve the specificity and affinity of naturally occurring and designed protein-protein interaction. Our approach recently resulted in the creation of a high-affinity, zinc-controlled protein homodimer.
Using rational design we engineered novel metal binding sites on the β1 domain of Streptoccocal Protein G (Gβ1) to generate a protein-protein interaction with high affinity and specificity. Metal coordination allows for focused surface redesign, allowing for the preservation of naturally occurring or designed interfaces. The interaction is stabilized by both metal coordination through histidine side-chains and hydrophobic residues at the designed interface. Complex formation and assembly was evaluating using size exclusion chromatography and X-ray crystallography. To accurately measure the molecular weight of the protein variants we have used size-exclusion chromatography with multi-angle light scattering (SEC-MALS). This technique first separates proteins based on size, then the molecular weight is determined from scattered light. The Gβ1 monomer and metal-controlled dimers had molecular weight of 6.2 and 12.2-13.1 kDa, respectively. Crystal structures of the dimeric proteins highlights zinc metal binding and coordination at the designed interface.
The overall goal is to exploit and explore key thermodynamic parameters that are essential for protein complex formation. We measured the binding affinity of the metal-mediated protein-protein interactions using analytical ultracentrifugation. The affinity determined for the homodimers were determined to be less than 30 nM. In addition to a series of homodimer complexes we also recently designed a heterodimeric binding pair. This variant also functions as a metal-controlled dimer. Multidimensional NMR is being used to determine the tertiary structure of this novel heterodimer.
Funding Agencies: Department of Defense (DOD), California Metabolic Research Foundation, CSU Annual Biotechnology Symposium (CSUPERB)
Brian Maniaci is an ARCS Scholar (2016-Present) and recipient of the Arne N. Wick Predoctoral Fellowship (2017-2018)
Poster #: 151
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: antibody structure, metalloantibodies, protein-DNA interactions
Project Title: Characterization of 2C10: an anti-double stranded DNA antibody
Author List:
Villaseñor, Citlayi; Undergraduate, Chemistry & Biochemistry, San Diego State University, Presenting Author
Luong, Sally; Undergraduate, Chemistry & Biochemistry, San Diego State University, Presenting Author
Wu, Yunjin; Graduate, Chemistry & Biochemistry, San Diego State University
Farokhi, Elinaz; Graduate, Chemistry & Biochemistry, San Diego State University
Huxford, Tom; Faculty, Chemistry & Biochemistry, San Diego State University
Abstract: Mammalian immune systems employ proteins called antibodies that target and counteract the effects of harmful pathogens. The 2C10 antibody, which was isolated from mice that exhibited Lupus-like symptoms, binds double-stranded DNA (dsDNA) as its antigen. Due to its sequence similarity with a well characterized metalloantibody, we hypothesize that DNA binding by 2C10 is a consequence of it being a divalent metal ion binding metalloantibody. We employed the Bac-to-Bac baculovirus-encoded recombinant protein expression system to produce 2C10 Fab fragments from Spodoptera frugiperda (Sf9) insect cells. Both open reading frames of the antibody heavy and light chains are contained within one pFastBacDual shuttle plasmid. Both chains contain N-terminal secretion signals and the heavy chain possesses a histidine-tag at its C-terminus for affinity purification. Upon transformation of the 2C10 shuttle plasmid into DH10Bac Escherichia coli, recombinant bacmid DNA containing the antibody sequences plus the genes encoding for a mature insect baculovirus formed via recombination. Sf9 insect cells were transfected with bacmid DNA to produce intact, recombinant baculovirus. Western blot confirmed expression of 2C10. However, the recombinant 2C10 remained trapped within the insect cells. Sequencing of the 2C10 plasmid revealed that one amino acid residue was missing. We introduced the missing arginine by mutagenesis. This resulted in successful secretion of the antibody Fab. These results suggest that the baculovirus/Sf9 system is well suited for production of recombinant antibody fragments. We are currently optimizing antibody Fab expression levels in Sf9 cell suspensions. We aim to apply fluorescence binding and gel mobility shift assays as well as x-ray crystallography to study the antibody-DNA complex to determine its binding mode. This project has been supported by a grant from the National Institute of General Medical Sciences of the National Institutes of Health: SDSU MARC U*STAR 5T34GM008303-29 as well as CSUPERB and the Doris A. Howell Foundation.
Poster #: 152
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Transmembrane Signaling, Receptor Biochemistry, Structural and Functional Bioinformatics
Project Title: Thinking Beyond the Transmembrane Box of Biogenic Amine GPCRs: Structural and Functional Hotspots
Author List:
Quinn, Caroline; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Abrol, Ravinder; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract: G protein coupled receptors (GPCRs) control diverse signaling pathways across the cellular membrane. Many GPCRs activated by the same ligand couple to different G proteins to trigger specific intracellular pathways. What causes this specificity is not well understood. The receptors contain conserved amino acids in their sequence, which may provide insight on why highly homologous GPCRs activated by the same signaling ligand couple to different G proteins. Evolutionary Trace (ET) is a bioinformatics tool that is used to rank the conservation of amino acids in a protein by looking at the phylogenetic tree of that protein’s family. For this study, we focused on the biogenic amine receptors activated by serotonin, adrenaline, dopamine, histamine, and acetylcholine. The receptors were grouped into same ligand families (activated by the same ligand) as well as into same G protein family (receptors that activate the same G protein). Conserved residues are seen in both ligand-specific and Gprotein-specific receptor families. These residues are not only found unsurprisingly in the transmembrane (TM) regions, but also in the highly divergent intracellular loops, extracellular loops, and the C-terminus domains of the receptors. These hotspot residues in the non-TM regions display conserved contacts across the respective families in available structures, suggesting their role as non-TM anchor residues critical for receptor structure stability. Some of these residues are also implicated for their functional roles as their mutation in specific receptors has been shown to affect downstream signaling. In addition, we found residues in both TM and non-TM regions that were conserved within Gprotein-specific receptor families, but were different across these families. This latter group of residues are potential G protein selectivity hotspots whose mutation could be used to test their specific roles in G protein selective signaling. Even though the non-TM regions are highly divergent across GPCRs, they possess conserved residues that are vital for receptor structure and function.
Poster #: 153
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: ambient particulate matter, atmospheric chemistry, reactive oxygen species
Project Title: Study of the chemical and signaling bases of ambient particulate matter induced oxidative stress in alveolar macrophages
Author List:
Olivas, Micah; Undergraduate, Chemistry, California State University, Fresno, Presenting Author, Nagel Award Nominee
Flores, David; Graduate, Chemistry, California State University, Fresno, Presenting Author
Waterston, Anthony; Graduate, Chemistry, California State University, Fresno
Hasson, Alam; Faculty, Chemistry, California State University, Fresno
Dejean, Laurent; Faculty, Chemistry, California State University, Fresno
Abstract: In recent years, concerns have arisen over the negative health effects caused by exposure to high concentrations of small atmospheric particulate matter (PM) in heavily-polluted areas—innate defense mechanisms in the upper airway fail to capture particles with a diameter less than 2.5 microns (PM2.5), permitting the smallest pollutants to generate an acute inflammatory response at the distal ends of the respiratory tract. However, by fixating on particle size, we accept a limited perspective as to the pathology of PM-borne illness, neglecting the possibility that other particle characteristics play a role in disrupting the respiratory microenvironment. Atmospheric pollution sampling campaigns conducted by our team in Fresno and Claremont, California showed a high degree of variability in the chemical composition of PM across locations and durations of filter collection. Consequently, we elected to study three commonly-occurring cyclic diones (9,10 phenanthrenequinone, 1,2 naphthoquinone, and 1,4 naphthoquinone) and two commonly-occurring transition metals (Cu2+ and Fe3+) for their propensity to generate oxidative stress within a cellular context. Using a DCF fluorescence-based cell population assay of alveolar macrophages (AM), our team has demonstrated that exposure to each of the selected chemicals induces a unique trend in the upregulation of reactive oxygen species (ROS) formation. Remarkably, linear regression of the experimental stress response in AM suggested that cell populations treated with prepared solutions of quinones on average reached a peak intracellular ROS level at comparatively lower treatment concentrations than did cells treated with prepared solutions of the two transition metals. Given the oxidative role of the quinone-derivative Coenzyme-Q in mitochondrial metabolism, we suggest that quinones contained in PM2.5 may interfere with the activity of the Electron Transport Chain, leading eventually to an increase in ROS production. We are currently testing this hypothesis through a flow cytometry-based approach, in which fluorescent dyes MitoSOX and DCFH-DA distinguish mitochondrial from cytosolic ROS. By sensing and discriminating between separate organellar ROS signals, our proposed methodology may provide an unprecedented window into the subcellular dynamics of common atmospheric pollutants and the respiratory-borne cancers, asthma, and cardiopulmonary complications they give rise to.
Poster #: 154
Campus: Cal Poly San Luis Obispo
Poster Category: Biochemistry
Keywords: Osmolytes, Protein stabilization, Differential Scanning Fluorimetry
Project Title: Ambient temperature stabilization of proteins for diagnostic applications
Author List:
Torgerson, Julie; Undergraduate, Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, Presenting Author
Baghdoyan, Sophie; Undergraduate, Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, Presenting Author
Jen, Jason; Undergraduate, Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo
Kim, Katie; Undergraduate, Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo
Takahashi, Rei; Undergraduate, Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo
Heying, Michael; Faculty, Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo
Wilkinson, Steven; Faculty, Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo
Abstract: The push for personalized medicine in recent years has been fueled by the rapid rate of protein and peptide biomarker discovery. A key challenge to the implementation of these discoveries into clinical settings has been the unreliability of biospecimen integrity during the time between sample collection and analysis. There currently exists no technological solution to the problem of protein biomarker denaturation ex vivo other than costly cold-chain logistics (ex. ice and refrigeration). An ideal solution to this challenge in molecular diagnostics would be the incorporation of highly soluble, small molecule stabilizers into biospecimen (ex. blood) collection devices that serve to maintain the structure and activity of protein biomarkers during the temperature extremes encountered during sample transport and storage. In this study, we compared the effects of fourteen small molecule “osmolytes” on the thermal stabilities of two diagnostically-relevant proteins, human tumor necrosis factor (TNFα) and human C-reactive protein (CRP). The stabilizing osmolytes in this investigation belong to three chemical classes: polyols and carbohydrates, amino acids and methylammonium salts. Using Differential Scanning Fluorimetry (DSF), we quantified protein melting temperature as a function of molar, and mass per volume, osmolyte concentrations. The osmolytes’ influence on thermal stability were compared by multiple metrics: 1) maximal effect on thermal stability and 2) m-values, as determined by measurements of Tm as a function of osmolyte concentration. These data reveal significant differences in the stabilization effects of the osmolytes under investigation. Moreover, the effects of these osmolytes are protein-specific, arguing against a simple hierarchal ranking of osmolytes. Therefore, we analyzed these data for potential correlations between stabilization effectiveness and physical and chemical properties of the osmolytes, such as chemical class and fractional polar surface area (FPSA). Our data clearly contradicts hypotheses from other groups that argue for a correlation between osmolyte effectiveness and FPSA. Such information is critical to the future development of broad spectrum, cold-chain-independent biospecimen stabilization chemistries.
(This project was supported by a CSUPERB New Investigator Award to Steven Wilkinson).
Poster #: 155
Campus: San Francisco State University
Poster Category: Biochemistry
Keywords: Dehydrogenase, Thioester, green chemistry
Project Title: Development of Assays for the Directed Evolution of Phenylacetaldehyde Dehydrogenase
Author List:
Jarvis-Jones, Kaila; Undergraduate, Chemistry and Biochemistry, San Francisco State University, Presenting Author
Abstract: Aldehyde dehydrogenases (ALDH’s) are responsible for oxidation reactions, esterase activity and in some cases, the synthesis of thioesters. In this work, we developed assays to evaluate the dehydrogenase, esterase and thioester synthetic capabilities of phenylacetalaldehyde dehydrogenase (PADH). PADH has a dehydrogenase mechanism catalyzing the NAD+-dependent oxidation of phenylacetalaldehyde (PAL) to phenylacetic acid (PAA) in the styrene metabolic pathway of P. putida (S12). Previously, we evaluated the kinetics of the transthioesterification reaction of PADH with aldehyde and nitrophenylester substrates in the presence of thiols as co-substrates. Thiol substrates were found to have reaction rates ranging from 4µMmin-1 to 30µMmin-1. We developed a method based on the Ellman Reagent for the detection and estimation of yield of thioesters with a limit of detection in the micromolar range. The assay indicated thioester products do not accumulate in this reaction and we conclude that the intrinsic esterase activity of PADH rapidly hydrolyzes the thioester products. This hypothesis was tested by using a carbodiimde-based organic synthesis to prepare a series of thioesters and and then using these compounds as substrates in steady state esterase activity assay with PADH. Our synthetic thioesters recovered in greater than 95% purity based on analysis by Ellman assay, thin layer chromatography and 1H NMR spectra. Using a steady state assay, we reacted various concentrations of our synthetic thioester with PADH. We then fit kinetic data from these reations. A second order rate constant of 0.725 ± 0.135 mM-1s-1 was determined for thioester hydrolysis in the PADH-catalyzed production of phenylacetic acid (PAA) and N-acetylcysteamine (SNAC). The assays developed in this work allow us to develop a structure-reactivity profile of PADH with aldehydes, thiols, and thioesters as substrates and provides the information we need to select PADH mutants modified by active site engineering that favor transthioesterification and inhibit thioesterase activity. Targeting thioesters derived from N-acetylcysteamine (SNAC) is of biotechnological value as they can be used with N-acyltransferases to produce intermediates in the production of pharmaceuticals like penicillin. Using the broad substrate specificity of PADH to construct SNAC derived thioesters will eliminate the use of toxic solvents, promoting green chemistry and create a simpler pathway to derive medicinal products.
Poster #: 156
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Peroxisome Proliferator Activated Receptors, Lipoprotein Lipase, ATP-Binding Cassette A1 Cholesterol Transporter
Project Title: Lipoprotein Lipase regulates ATP-Binding Cassette A1 transcription independently of Peroxisome Proliferator-Activated Receptors
Author List:
Christian, Bryan; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Moctar, Khadija; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Medh, Jheem; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract: Introduction: Several factors regulate macrophage cholesterol content. Lipoprotein lipase (LPL) promotes cellular lipid uptake. ATP-Binding Cassette A1 (ABCA1) facilitates cholesterol efflux from cells, and Peroxisome Proliferator-Activated Receptors (PPAR) are nuclear receptor transcription factors that regulate expression of genes relevant to lipid metabolism. In previous studies, we demonstrated an upregulation of ABCA1 in LPL-knock-down (KD) THP-1-derived macrophages.
Hypothesis and Objective: We hypothesized that fatty acid products of lipolysis may act as agonists/antagonists for PPARs and thereby modulate ABCA1 expression. Thus, the objective of this study was to determine if the upregulation of ABCA1 by LPL-silencing may be via a PPAR-dependent pathway.
Methods: Short hairpin RNA was used to specifically silence the LPL or PPAR-γ gene. End-point reverse transcriptase polymerase chain reaction (RT-PCR) was used to compare gene-specific transcript levels in wild-type (WT) cells and either LPL-KD or PPAR-γ-KD macrophages. The effects of fatty acids (products of lipolysis) on the binding of PPAR to PPRE was examined by ELISA. A PPAR-luciferase reporter gene assay compared the PPAR-activating potential of lipid extracts from WT or LPL-KD macrophages. Western blot analysis was used to examine if fatty acid products of lipolysis alter ABCA1 protein expression.
Results: RT-PCR showed that the level of PPAR-γ transcripts in LPL-KD cells was 93% of that in WT cells. PPAR-γ-KD cells showed a down-regulation of LPL mRNA to 27% of WT levels, and a repression of ABCA1 transcription to only 1% of WT levels. Saturated and monounsaturated fatty acids significantly decreased PPAR binding to PPRE. However, Luciferase assay data showed that lipid extracts from LPL-KD macrophages exhibited lower PPAR activation than WT lipid extracts, thus suggesting that fatty acid products of lipolysis may function as co-repressors of PPAR-mediated transcription. Consistent with this observation, fatty acids did not significantly alter ABCA1 protein expression levels.
Conclusion: Our data demonstrate reduced PPAR-γ transcriptional activity in LPL-KD cells. Since PPAR- γ was found to induce ABCA1 transcription and function, it is clear that ABCA1 up-regulation in LPLKD cells is independent of PPAR- γ.
Acknowledgements: This work was supported by NIH Award SC3GM095413.
Poster #: 157
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: DNA Origami, Nanostructures, Bioengineering
Project Title: Nanoscale Design Utilizing DNA Folding Techniques
Author List:
Serrano, Allison; Undergraduate, Biological Sciences, California State University, Fullerton, Presenting Author
Alvarez-Loya, Axel; Undergraduate, Mechanical Engineering, Presenting Author
Arroyo, Daniel; Undergraduate, Biological Sciences, California State University, Fullerton
Fitchett, Noelle; Undergraduate, English Department, California State University, Fullerton
Chen, Binyun; Graduate, Mechanical Engineering, California State University, Fullerton
Fayard, Samuel; Undergraduate, Foothill Community College
Robson, Nina; Faculty, Mechanical Engineering, California State University, Fullerton
Rasche, Madeline; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Abstract: DNA Origami, a relatively new application in scientific research, is a technique featuring the self-assembly of DNA template strands and primers for the formation of shapes, patterns, and nanoscale molecular machines. In the current work, a cross-disciplinary team of mechanical engineering and biological science students have adapted the DNA Origami protocols of Rothemund (2006) for the design of simple DNA origami structures using the equipment present at CSU Fullerton (CSUF). Because the existing thermocycler programs could not accommodate the speed and continuity of the published temperature gradient, the CSUF thermocycler was reprogrammed in an effort to anneal the primers to Viral Template DNA (M13mp18) with a steady decrease in temperature of 1°C/min in 0.1 degree increments. Verification of the DNA complex formation was performed by running samples through a 1% agarose gel. Relative to the band for linear M13mp18 template alone, a discrete band for the DNA origami structure was shifted to a higher molecular weight, indicating successful template-primer binding to produce the desired structure. Following completion of the initial DNA Origami structure, the team was inspired to create an original DNA Origami design: a CSUF logo consisting of a circular shape with the letter F in the center (circle F), formed by absences of DNA helical structure. An initial circle F design was engineered using the CAD-based program CaDNAno paired with Autodesk Maya 2015, which produced a three-dimensional image of the design created. The primers were then manually refined to increase binding stability of the 175 primers proposed by the CaDNAno software. The successful adaptation of DNA Origami protocols to the resources available at CSUF demonstrate the potential and proof-of-concept for CSU researchers to both design and create DNA nanostructures, which may have applications to solving biological problems such as molecular sorting and drug delivery in the future.
Reference: Rothemund, P.W.K (2006) Folding DNA to create nanoscale shapes and patterns. Nature 440:297-302
Acknowledgements: The authors gratefully acknowledge the support of 2017 CSUPERB Curriculum Development Grant Program and NSF CAREER grant #1751770.
Poster #: 158
Campus: CSU East Bay
Poster Category: Biochemistry
Keywords: Oligonucleotide, Imaging, Contrast agents
Project Title: Development of aptamer-based contrast agents with lower toxicity
Author List:
Kadeer, Elshad; Graduate, Chemistry and Biochemistry, California State University, East Bay, Presenting Author
Heidarian, Mohammad; Undergraduate, Chemistry and Biochemistry, California State University, East Bay, Presenting Author
Halim, Marlin; Faculty, Chemistry and Biochemistry, California State University, East Bay
Abstract: In clinical magnetic resonance imaging (MRI), contrast agents (CAs), which serve to enhance the resolution of the images obtained, are often administered to the patients undergoing the procedure. One commonly used agent is a class of compounds called gadolinium-based CAs (GBCAs). However, although effective, they have been implicated in the development of nephrogenic systemic fibrosis, a disorder that may severely affect the proper functioning of some organs.
Our goal is to develop a GBCA with a higher efficacy. We hypothesize that this improvement will allow for reduced dosage and therefore, there will be a lower concentration of the agent that can cause the adverse side effects.
The efficacy of a GBCA is size-dependent; it increases with a higher molecular weight of the agent. The current clinical GBCAs are ~0.5 kDa in size and consist of a Gd3+-organic ligand complex, i.e. a Gd3+ ion chelated by an organic molecule. We proposed a design where we encapsulate this traditional GBCA complex with an aptamer. The aptamer is a short oligonucleotide (~50 bases long) that can bind tightly and selectively to the GBCA. This will then significantly increase the size of the CA from ~0.5 to ~17 kDa.
The first step in the experiment was to prepare the Gd3+-organic ligand complex using organic synthesis techniques. Through a series of nucleophilic substitution and metal complexation reactions, the purified final compound was obtained in ~80% yield over 3 steps. The products were characterized using nuclear magnetic resonance spectroscopy and mass spectrometry, which confirmed the success of the synthesis. The next step was to isolate an aptamer with a high affinity and selectivity for the synthesized GBCA. To achieve this, an in vitro selection procedure that has been previously established in our group, is employed. This process is iterative and at the end of each cycle, aptamers with successively better binding parameters to the GBCA are produced. We have performed 10 selection cycles and characterization via gel electrophoresis, ethidium bromide staining, and fluorescent imaging led us to conclude that all the experiments were successful. The most recent iteration produced 36 ng/uL of aptamers (as measured via UV-VIS absorption) that could bind to 80 uM of GBCA.
Future work is to continue the selection to obtain aptamers that can bind to 10 uM of GBCA. Subsequently, the efficacy of the GBCA-aptamer agent will be quantified on a magnetic resonance spectrometer.
Poster #: 159
Campus: CSU San Bernardino
Poster Category: Biochemistry
Keywords: Lipid metabolism, Peroxisome, Cell signaling
Project Title: Determining the physiological significance of a Rab GAP (GTPase accelerating protein) found at peroxisomes in yeast
Author List:
Mora, Adrian; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Delgado, Beatriz; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Nickerson, Daniel; Faculty, Biology, California State University, San Bernardino
Abstract: Rab GTPases (Rabs) are lipid-anchored proteins that regulate membrane docking and fusion of vesicles and membrane-bound organelles. Rabs change activity based on their association with GTP (active) or GDP (inactive), however, the rate at which GTP is hydrolyzed into GDP is slow. Rabs require accessory proteins called Rab GAPs (GTPase accelerating proteins) to accelerate GTP hydrolysis and inactivate Rab signal. We have identified a Rab GAP, Gyp8, that localizes to peroxisome membranes in the yeast S. cerevisiae. Peroxisomes are membrane-bound organelles that serve several functions, including using reactive oxygen species to break down toxic waste products and fatty acids. This is the first report in any experimental system of a Rab GAP localized to peroxisomes, which demands an investigation of possible physiological roles of Gyp8 in regulating peroxisome dynamics.
Cells lacking Gyp8 suffer no overt defects in peroxisome function or cargo transport, as fluorescence microscopy indicates that gyp8 mutants possess peroxisomes that correctly localize peroxisome cargo proteins that must be imported from the cytosol. Studies of peroxisome cargo import using sensitive, quantitative colorimetric assays are ongoing.
Competitive growth assays revealed that cells lacking Gyp8 suffer a disadvantage compared to wild type cells when grown in peroxisome-stressing media, but not when grown in unstressed conditions. To examine possible roles of Gyp8 in peroxisome creation or destruction, cells were monitored when fed oleic acid media that requires peroxisome function for cell viability and growth. Cells lacking Gyp8 were indistinguishable from wild type cells during the early phases of growth in oleic acid media, suggesting that Gyp8 is not essential for peroxisome biogenesis and fatty acid breakdown. However, cells lacking Gyp8 saturated their growth at a lower density than wild type cells, producing microbial growth curves that suggested a lack of diauxic shift or other functional adaptation when all oleic acid had been consumed. When oleic acid is depleted, yeast are known to rapidly destroy their surplus peroxisomes. We hypothesize that cells lacking Gyp8 may suffer slower kinetics of peroxisome destruction via pexophagy. Ongoing studies address the kinetics of pexophagy using luciferase reporter assays in the presence, absence and overabundance of Gyp8.
This work was supported by a NIH MARC fellowship and SURP grant from CSUSB OSR.
Poster #: 160
Campus: CSU San Bernardino
Poster Category: Biochemistry
Keywords: Membrane transport, Cell signaling, Quality control
Project Title: Gyp8 is a membrane-anchored Rab GAP (GTPase accelerating protein) whose correct targeting to peroxisomes depends upon chaperone Msp1
Author List:
Quinn, Monique; Graduate, Biology, California State University, San Bernardino, Presenting Author
Lin, Ariel; Undergraduate, Chemistry, California State University, San Bernardino, Presenting Author
Nickerson, Daniel; Faculty, Biology, California State University, San Bernardino
Abstract: Eukaryotic cells use vesicular transport pathways to build and maintain membrane-bound organelles that house specific biochemical functions. Rab GTPase signaling proteins (Rabs) are key regulators of vesicular transport, controlling where and when membranes can dock and fuse, delivering lipid and protein contents to appropriate destinations. Rabs adopt their signaling active conformation when bound to GTP, which allows Rab interactions with tethering and membrane fusion proteins. Rab proteins have a slow intrinsic rate of hydrolyzing GTP, so Rabs depend upon GTPase accelerating proteins (GAPs) to trigger GTP hydrolysis and return the Rab to its GDP-bound, inactive state. Rab GAPs are important to regulate the efficiency and fidelity of transport pathways, and defective Rab GAPs are implicated in a variety of human diseases. Rab GAPs tend to be cytosolic proteins that briefly localize to membranes to survey for their client Rab. We report that the yeast Rab GAP Gyp8 is an unusual transmembrane (TM) GAP whose association with peroxisomes is regulated by the ATPase Msp1, a chaperone that functions to remove tail-anchored proteins from peroxisomes and mitochondria. Fluoresence microscopy indicated that GFP-tagged Gyp8 co-localized with peroxisomal markers in wild type cells. Computational analysis of Gyp8 predicts a single-pass TM domain near the carboxy terminus, characteristic of a tail-anchored protein. Loss of Msp1 chaperone function resulted in mislocalization of GFP-Gyp8 to non-peroxisomal membranes. In the absence of peroxisomes, GFP-Gyp8 redistributed to the endoplasmic reticulum (ER). Subcellular fractionation biochemically demonstrated that Gyp8 localized exclusively to a membrane fraction containing peroxisomes. Truncation analysis of Gyp8 indicated that the TM and luminal domains are necessary and sufficient to direct localization to peroxisomes. Loss of the luminal domain resulted in GFP-Gyp8 localizing to non-peroxisomal membranes. Ongoing studies will identify by co-localization the specific non-peroxisomal membranes (putatively mitochondria and ER) to which GFP-Gyp8 mislocalizes, as well as biochemically verifying that Gyp8 is an integral membrane protein. These data represent the first report in any experimental system of a Rab GAP that localizes to peroxisomes. Further study will explore the presumptive physiological role of Gyp8 in regulating peroxisome dynamics. This work was supported by SURP and student research grants from CSUSB OSR.
Poster #: 161
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: heat-shock proteins, lipids, mutations
Project Title: HspA1A, a 70-kDa heat-shock protein, contains several distinct lipid-binding sites
Author List:
Daniels, Amanda; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Briseno, Carolina; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Nikolaidis, Nikolas; Faculty, Biological Science, California State University, Fullerton
Abstract: HspA1A, a stress-inducible seventy-kilodalton heat shock protein (Hsp70), is a molecular chaperone that plays critical roles in protein homeostasis and cellular survival. In addition to its functions in protein homeostasis, HspA1A also localizes at different cellular membranes in stressed and cancer cells, where it binds to lipids, including phosphatidylserine (PS) and Bis(Monoacylglycero)Phosphate (BMP). Although these interactions have significant physiological consequences, their mechanistic details remain elusive and unexplored, because the amino acids responsible for the lipid-binding remain largely unknown. Here, we further characterized the properties of a mutation from Tryptophan to Phenylalanine (W90F), which has been described to affect the binding of HspA1A to BMP. We first determined whether this mutation alters HspA1A’s stability, intracellular and membrane localization, as well as its ATPase and refolding functions using recombinant proteins and ex vivo experiments. Our results revealed that the W90F mutation does not significantly alter any of the tested functions. We then used the lipid vesicle sedimentation (LVS) method and Surface Plasmon Resonance to quantify the binding of the wild-type (WT) and W90F-HspA1A to both PS and BMP. These experiments revealed that although the W90F-HspA1A binding to BMP was different than the WT, this mutation did not affect the binding to PS. Together these results strongly support the prediction that the lipid-binding sites for PS and BMP are distinct and lipid-specific. To further support this prediction, we used a combination of structural superimpositions, sequence alignments, and literature observations and predicted three putative lipid-binding regions on the HspA1A molecule. We tested these predictions using the LVS assay for 20 single or double mutations spanning two of these regions. Our results revealed that two of these mutations affect the binding to PS, while a different one minimizes the binding to BMP. These findings provide further evidence that HspA1A binds to PS and BMP using different amino acid sites and will allow us to test the effects of these mutations on the membrane-localized functions of HspA1A in cell lines and cancer cells.
Poster #: 162
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: NMR , Nanoparticles, Drug Delivery
Project Title: Elucidating the Molecular Structure, Dynamics and Assembly of Amino Acids and Peptides at Nanoparticle Interfaces
Author List:
Cao, Michael; Undergraduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Swanson, Haley; Graduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Addison, Bennett; Staff, Department of Chemistry and Biochemistry, San Diego State University
Holland , Gregory; Faculty, Department of Chemistry and Biochemistry, San Diego State University
Abstract: Nanoscience has become a major research area because of its incredible potential for applications in biotechnology. A range of nanosystems has been explored for drug delivery applications including: nanoparticles (NPs), nanofibers, polymer micelles, carbon nanotubes, viral vectors, among others. Due to their small size, NPs have the unique ability to penetrate cells. They form dynamic entities when placed in biological media and can incorporate a diverse range of features which can be manipulated to induce targeted drug release upon external stimuli such as heat or pH. In addition to drug delivery and cancer therapeutics, nanomaterials are also used to enhance the biocompatibility of sensors, detectors, and other medical technological devices. However, it is not well understood how biomolecules such as proteins and peptides interact, bind and assemble at the nanomaterial interface. Our current work seeks to answer some of these questions by understanding how individual amino acids interact with silica NP interfaces and how the structure of these amino acids may change when exposed to heat. Specifically, we are investigating the polymerization of alanine and lysine on fumed silica NPs. We hypothesize that this type of silica nanoparticle acts as a catalyst and converts alanine to its cyclized form, alanine anhydride, following mild heat treatment. We verified that alanine undergoes thermal condensation through thermogravimetric analysis (TGA). Then we used solution state nuclear magnetic resonance (NMR) methods including: 1H, 13C, HSQC, and HMBC, to confirm the resulting structure of alanine after thermal condensation. Preliminary results on lysine indicate that one product is preferentially formed after exposure to heat, as evidenced by a single peak in the peptide condensation region of the TGA curve. On a multiplicity edited HSQC, there were two CH2 peaks which can be attributed to free or unreacted lysine and a lysine peptide. Further characterization will include high performance liquid chromatography (HPLC) and matrix assisted laser desorption/ionization (MALDI) to aid in structural characterization of the lysine peptide. We anticipate that these results can provide an approach to fabricate nanomaterials with biomolecular functionalization and provide a better understanding of the molecular structure, assembly, and chemistry of amino acids and peptides on NPs.
Poster #: 163
Campus: Sonoma State University
Poster Category: Biochemistry
Keywords: Polysulfur, Phosphatase, Persulfide
Project Title: Cysteine Trisulfide Protects Cells Against Electrophile-Induced Death
Author List:
Henderson, Catherine; Undergraduate, Biology, Sonoma State University, Presenting Author
Long, Faith; Undergraduate, Biology, Sonoma State University, Presenting Author
Fukuto, Jon; Faculty, Chemistry, Sonoma State University
Lin, Joseph; Faculty, Biology, Sonoma State University
Abstract: Polysulfur compounds (RSSn R, n > 1) have long been thought to protect cells from oxidative damage, however the mechanisms by which this occurs remains unclear. Some believe that the protective effects seen from polysulfur compounds are because cells can metabolize them to generate hydrogen sulfide (H2S). H2S has been implicated as an endogenous signaling agent with broad physiological effects. We believe that H2S in the presence of oxidized thiols (RSSR) or polysulfur compounds, exist in equilibrium with another chemically relevant sulfur species, persulfides (RSSH). These persulfide species are stronger nucleophiles than their corresponding thiols and are generated by cells at biologically relevant concentrations. The full biological implications of these various sulfur species are not yet known but persulfidation of cysteine residues has been shown to have an impact on protein activity on a wide variety of proteins.
Here, we show that the treatment of HEK 293T cells with the polysulfide cysteine trisulfide (Cys-SSS-Cys) results in significantly higher intracellular concentrations of cysteine persulfide (Cys-SSH). Significantly, when cells pre-treated with cysteine trisulfide are then challenged with the electrophile NEM (N-ethylmaleimide), the cells are protected from cell death. Since NEM-induced cell death is likely due to oxidation of critical cellular thiols, we chose to examine CD148, a cysteine dependent protein tyrosine phosphatase.
To initially characterize CD148, we purified recombinant protein and measured its sensitivity to electrophiles. We found that CD148 could indeed be inactivated by NEM (IC50 at 15 min: 146.6 mM ± 2.62)). CD148 has cysteines that are non-catalytic but presumably play a regulatory, protective, or structural role in protein function. To determine what role noncatalytic cysteines play, we mutated all of the noncatalytic cysteines to serines, leaving only the catalytic cysteine (4C/S). Interestingly, the 4C/S mutant had dramatically increased sensitivity to NEM inactivation (IC50 at 15 min: 0.434 mM ± 0.13) indicating a protective role for the non-catalytic cysteines. CD148 was then expressed in HEK 293T cells and the cells subjected to NEM treatment. The 4C/S mutant again showed increased sensitivity to NEM treatment in cells, supporting a protective role for non-catalytic cysteines. Currently, we are investigating whether cysteine trisulfide pretreatment protects CD148 from NEM inactivation.
Poster #: 164
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: ceruloplasmin, canines, copper
Project Title: Investigation of Canine Ceruloplasmin – Why is it acting like a much larger protein than it is?
Author List:
Alam, Yasmine; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author, Nagel Award Nominee
Kim, Kaitlynne; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Linder, Maria C; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Abstract: Copper is an essential trace element found in all living organisms. It is necessary in mammals for processes and enzymes that range from electron transport in mitochondria to antioxidant defenses. Previous research found that dogs have 10-20 times higher copper concentrations in the liver than do other mammals, and this is accompanied by a tendency to develop copper overload. We have further discovered that canine blood plasma has peculiar characteristics regarding the main copper binding protein, ceruloplasmin (Cp). Specifically, upon fractionation of plasma through large pore size exclusion chromatography (SEC) (Superdex 200) equilibrated with 20 mM phosphate (pH 7), copper profiles of several canine breeds indicated that Cp elutes much earlier than that of other mammals, indicating it is acting as if much larger. The goal of my research is to determine whether this is due to binding another protein(s) (which has been reported to occur in some conditions), or to aggregation. SDS-PAGE-Western blotting, and assays of ferroxidase activity (a characteristic of Cp) showed that Cp polypeptide was of normal size and function. Further SEC studies used gravity flow and an even larger pore resin (Sephacryl S300), in the same buffer as well as in physiological phosphate-buffered saline. Here, the canine Cp either still eluted early or dissociated into two (still large) peaks of copper and ferroxidase activity. Stained SDS-PAGE gels of the Cu peak(s) fractions did not show any obvious bands with the same elution pattern as Cp to which Cp might be binding. When the SEC was carried out in 300mM K phosphate (pH 6.8) however, the Cp peak eluted much later, and in the same way as Cp of other mammals. The Cp of a fox (which belongs to the same biological family as dogs; Canidae), behaved like that of other mammals and not dogs. First attempts to detect other binding proteins by co-immunoprecipitation of dog Cp (eluting as large material) did not show Cp bands on stained gels, probably due to the non-affinity purified antibody. Further analyses by Western blotting and with more appropriate antibody are planned. Analysis of potentially unique features in canine Cp amino acid sequence has begun. Although still preliminary, the results suggest that canine Cp is aggregating with itself rather than binding to another protein, the chemical basis of which remains to be determined.
Poster #: 165
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: Thermotoga maritima, Kinase, tRNA modification
Project Title: The role of kinase activity in the biosynthesis of threonylcarbamoyl adenosine in bacteria
Author List:
Paranagama, Naduni; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Luthra, Amit; Postdoc, Chemistry and Biochemistry, San Diego State University
Bayooz, Susan; Staff, Chemistry and Biochemistry, San Diego State University
Swairjo, Manal; Faculty, Chemistry and Biochemistry, San Diego State University
Abstract: tRNA, the central molecule of translation, is heavily modified posttranscriptionally with noncanonical nucleosides that constitute ~83% of all RNA modifications. These modifications cluster in the anticodon stem loop (ASL) and core regions (D- and T-loops) of tRNA, and they are necessary for translational fidelity and diversification of codons. The ASL modification N6-threonylcarbamoyl adenosine (t6A) found at position 37 in ANN-decoding tRNAs (N is any nucleobase) is universally conserved in all kingdoms of life. Due to its essentiality in bacteria, the t6A biosynthesis pathway has recently emerged as a potential antibacterial target. Bacterial t6A biosynthesis begins with synthesis of the intermediate threonylcarbamoyl adenylate (TC-AMP) from threonine, ATP and bicarbonate by the TsaC enzyme, followed by transfer of the threonylcarbamoyl moiety from TC-AMP to A37 of substrate tRNA by the theonylcarbamoyl transfer (TCT) complex, comprised of the three proteins TsaB, TsaD and TsaE. We recently showed in T. maritima that while the TsaBD complex is the substrate binding platform responsible for TC transfer, the complex becomes inactivated after each t6A synthesis cycle, and a TsaE-dependent ATP hydrolysis step “resets” the platform for multi-turnover catalysis. This led us to propose that ATP hydrolysis affects a chemical change such as phosphorylation that occurs in the TCT complex after TC transfer. To test this hypothesis, we investigated the kinase activity of TsaE using a combination of radiochemical assays, X-ray crystallographic and mutagenesis experiments. The results show that in the free form, TsaE acts as a tyrosine kinase that autophosphorylates at the invariant surface residue Tyr82. When bound in the TCT complex, kinase activity is abolished due to sequestration of Tyr82 in a tight pocket in TsaD. Further, all active site mutations that abolished kinase activity also abolished ATPase activity, but not vice versa, indicating that the two activities are independent of each other and suggesting a putative transphosphorylation/phosphatase activity of the TCT complex. Further investigation of this hypothesis is underway.
Funding: NIGMS grant GM110588 to M.A.S.; California Metabolic Research Foundation; U.S. DOE grant DEAC02–76SF00515; NIGMS grant P41GM103393; SDSU, CSUPERB.
Poster #: 166
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: Alternative splicing , unstructured regions, phosphorylation
Project Title: Determining the role of linker region phosphorylation in Polypyrimidine Tract Binding Protein 2 splicing activity
Author List:
Hitch, Elizabeth; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Madory, Dean; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Keppetipola, Niroshika; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Abstract: The Polypyrimidine Tract Binding Protein 1 (PTBP1) is an RNA binding protein that controls the alternative splicing of many gene transcripts. PTBP1 belongs to a gene family with paralogs expressed in varying cell types. PTPB1 is expressed in most cell types but is absent in differentiating neurons and muscle. Paralog PTBP2 is highly expressed in differentiating neurons. The amino acid sequence of PTBP1 is 74% identical to PTBP2 and the two proteins have a similar domain organization with 4 RNA binding domains connected by 3 linker regions and an N-terminal region. The two proteins have over-lapping and non-overlapping target exons; PTBP1 functions as a splicing repressor of many neuronal exons while PTBP2 does not. PTBP1 is expressed in neuronal progenitor cells, but is down-regulated during neuronal differentiation while the level of PTBP2 is up-regulated. The switch in the levels of the two proteins effects the splicing of many exons that are sensitive to PTBP1, thus causing a change in the neuronal splicing program, that is critical for the development and maturation of neurons. How the two proteins exert different splicing outcomes is not well understood. Previous studies in the lab indicate that PTBP1 and PTBP2 are post-translationally modified by phosphorylation and that PTBP2 has many more non-overlapping sites of phosphorylation than PTBP1. Moreover, PTBP2 distinct phosphorylation sites are located in the unstructured N-terminal, linker 1 and linker 2 regions that can influence protein-RNA and protein-protein interactions. Thus, we hypothesize that phosphorylation of PTBP2 N-terminal and linker regions dictates its neuron-specific splicing activity. To test this, we created PTBP1-PTBP2 chimeric proteins containing either PTBP1 N-terminal, Linker 1, or Linker 2 regions in an otherwise PTBP2 protein via two-step PCR. The PCR products were digested and ligated into a mammalian expression vector and sequenced to verify the absence of unwanted coding changes. The chimeras were assayed for protein expression in vivo in mouse neuro2A cells using Western Blot. Our data highlight the chimeras are well expressed. We are currently conducting in vivo splicing assays using reporter mini genes that contain PTBP1 repressed exons to assay splicing activity of the chimeras. Collectively, results from this study will reveal the role of the N-terminal, Linker 1 and Linker 2 region in PTBP2 neuron-specific splicing activity.
Poster #: 167
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Biochemistry, DNA Repair, Molecular Cloning
Project Title: Construction of Plasmids Encoding Fluorescently-labeled RAD10 and YEN1 to Study Temporal and Spatial Relationships during DNA Repair in S. Cerevisiae
Author List:
Kalkatechi, Sahel; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Hadi, Hafridha; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Fischhaber, Paula; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract: DNA damage sites, such as double-strand breaks (DSBs), are considered mutagenic because they cause alterations in DNA sequence that can give rise to cancer if left unrepaired. Endonucleases including the Rad1-Rad10 complex and Yen1 participate in the final steps of DSB repair. Both endonucleases recognize DNA in a structure-specific fashion with the Rad1-Rad10 complex cleaving Y-shaped single-strand, double-strand junctions and 3′-flapped DNAs, while Yen1 recognizes joint molecules (Holliday Junctions) and cleaves them, thereby separating intertwined chromosomes during the final steps of repair. Yen1 acts in a cell-cycle dependent manner, carrying out most of its function in anaphase. Rad1-Rad10 is believed to function in all phases of the cell cycle but could be more active in some than others.
Our aim is to investigate the timing of action of Rad10 and Yen1 in DSB repair in Saccharomyces cerevisiae by fluorescence microscopy during which we will fluorescently label each gene and monitor recruitment of protein products to induced DSB sites. Prior work with fluorescently-labeled RAD10 (Rad10-YFP) gave dim images with low signal-to-noise due to low abundance of Rad1-Rad10 in the cells. Additionally, Yen1 appears to be in lower abundance relative to other proteins present during anaphase, and therefore, likely to suffer from the same low signal-to-noise issue. In this project, we are constructing labeled genes (RAD10-3xYFP and YEN1-EmRFP) by installing three tandem Yellow Fluorescent Protein labels at the C-terminus of RAD10 and a bright version of Red Fluorescent Protein at the C-terminus of YEN1. To construct these strains, we began by preparing DNA plasmids containing sequence coding for the fluorescent labels, partial URA3 selection markers, and fragments of the yeast chromosomes representing the sites destined for label integration. Using adaptamer-mediated Polymerase Chain Reactions and DNA ligations, we have cloned the DNA plasmids that will enable integration of RAD10-3xYFP into yeast. Completed DNA plasmids were characterized by restriction digestions and Polymerase Chain Reaction assays. Further, we have created several DNA fragments needed for the Yen1-EmRFP plasmids as characterized by agarose gel electrophoresis. Together these DNA plasmids will allow cloning of yeast strains that will be powerful research tools providing increased signal-to-noise, and thereby broadening the variety of experiments that can be conducted on Rad10 and Yen1.
Poster #: 168
Campus: Cal Poly San Luis Obispo
Poster Category: Biochemistry
Keywords: Kinases, Expanded genetic code, MAPK Pathway
Project Title: Enabling the enzymology of human kinases through an expanded genetic code
Author List:
Vojvoda, Elizabeth ; Undergraduate, Chem/Biochem, California Polytechnic State University, San Luis Obispo, Presenting Author, Nagel Award Nominee
Ravatt, Leandre; Undergraduate, Chem/Biochem, California Polytechnic State University, San Luis Obispo
Lee, Rebecca; Undergraduate, Chem/Biochem, California Polytechnic State University, San Luis Obispo
Runco, Caroline; Undergraduate, Chem/Biochem, California Polytechnic State University, San Luis Obispo
Oza, Javin; Faculty, Chem/Biochem, California Polytechnic State University, San Luis Obispo
Abstract: The human Mitogen-Activated Protein Kinase (MAPK) signal transduction cascades are prevalent cell proliferation signaling pathways implicated in 90% of cancerous melanomas and numerous other diseases. The MAPK pathways share a defining characteristic: the interactions between an active, phosphorylated kinase of the MAP2K kinase family and its substrate, such as MEK1 and its substrate ERK2, create a bottleneck in each cascade that all signals pass through, making members of the MAP2K kinase family ideal targets for therapeutic intervention. The characterization of MAP2K kinases has been limited due to the technical challenge of obtaining homogeneous samples of site-specifically phosphorylated kinases for enzymology. While phosphomimics of MEK1 are field-accepted forms of active MEK1, our preliminary evidence suggests that phoshphomimics do not activate the enzyme to the same extent as phosphorylation. We aim to overcome the limitation by utilizing an expanded genetic code to site-specifically install phosphoserine into MEK1. We are pursuing a hypothesis-driven, structure-guided approach based on conserved kinase motifs to investigate the structure-function relationships within MEK1. We have identified 16 residues in the catalytic and P+1 loops of MEK1 which we hypothesize are involved in substrate (ERK2) binding and phosphorylation. To date, we have created 15 MEK1 variants with single residue mutations to alanine using site-specific mutagenesis and have purified stocks of inactive, unphosphorylated MEK1, a phosphomimic of MEK1, one catalytic loop mutant of inactive MEK1, and ERK2. To assay the activity of MEK1 and its mutants, we are using a high-throughput immunoassay platform to quantify product formation – phosphorylated ERK2. Our preliminary assays have demonstrated that our activity assay is viable for the quantification of phosphorylated ERK2 product over time, enabling the characterization of MEK1 enzyme kinetics. Going forward, our work will be the first to establish kinetic parameters for the site-specifically phosphorylated human MEK1. We will then characterize the kinetics of active MEK1 mutants to identify residues vital for substrate binding and catalysis. Given the similarities between MAP2K kinases, a better understanding of the enzymology of MEK1 will provide insights into all other MAP2K kinases for mechanism-based drug design. Our research was funded by the Center for Applications in Biotechnology and the Bake-Koob Endowment.
Poster #: 169
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: kinetics, cancer metabolism, IDH1
Project Title: IDH1 Allosteric Inhibitors’ Potency Vary Depending on the Enzyme’s Native Activity
Author List:
Matteo, Diego Avellaneda; Graduate, Chemistry/Biochemistry, San Diego State University, Presenting Author
Luna, Lucas A.; Graduate, Chemistry/Biochemistry, San Diego State University
Schiffer, Jamie M.; Schrodinger
Sohl, Christal; Faculty, Chemistry/Biochemistry, San Diego State University
Abstract:
Isocitrate dehydrogenase 1 (IDH1) catalyzes the formation of α-ketoglutarate (αKG) from isocitrate. Mutations at residue 132 drive ~80% of lower grade gliomas and secondary glioblastomas. Most mutations ablate wildtype (WT) activity and facilitate a new neomorphic function – the reduction of αKG to D-2-hydroxyglutarate (D2HG). D2HG competitively inhibits many αKG-dependent enzymes responsible for gene regulation, and the FDA has recently approved a selective inhibitor for mutant IDH1. There is a wide structural and chemical diversity of the R132 mutants that drive tumorigenesis, but the catalytic features of these mutations are not well understood. We have shown previously that the tumor-relevant mutant R132Q IDH1 retains its WT activity, and here we hypothesize that mutant IDH1 inhibitors will not effectively ablate D2HG production by this mutant. To test this, we heterologously expressed and purified IDH1 mutants found in tumors and tested the activity of mutant IDH1 inhibitors. In vitro biochemical inhibition assays with AGI-5198 showed that the IC50 values for WT and R132Q IDH1 were similar (1000 µM and 650 µM, respectively), while the IC50 for R132Q IDH1 was >16,000-fold higher than that measured for R132H IDH1. Similar results were found for the inhibitor ML309, where a 125-fold increase in IC50 was measured for R132Q IDH1 compared to R132H IDH1. Only GSK864, which inhibits WT IDH1 at higher concentrations, was able to inhibit R132Q IDH1, though not as effectively as R132H IDH1. We used molecular dynamics simulations to create a model of R132Q IDH1 to determine the mechanism of its observed experimental catalytic and inhibitory properties. Simulations show that R132Q IDH1 fluctuates between two major conformations, a WT and a mutant-like conformation, suggesting a mechanism for the retained WT and mutant activities. Loss of coordination between a regulatory α10 helix and residue 132 appears crucial for opening an inhibitor binding pocket, and we propose that R132Q IDH1 is able to at least partially maintain this interaction to ablate inhibitor binding. Importantly, this work identifies IDH1 mutations that may be refractory to current clinical therapies, setting a crucial foundation for understanding how to effectively inhibit a wide panel of tumor-relevant mutations. We also highlight possible routes of resistance that may occur in patients following treatment with these targeted therapies.
Poster #: 170
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: Spider Silk, NMR, Biomaterials
Project Title: Water-Induced Changes in Molecular and Mechanical Properties of Spider Silk
Author List:
Trescott, Samantha; Undergraduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Alabdali, Rawan; Undergraduate, Department of Chemistry and Biochemistry, San Diego State University
Onofrei, David; Graduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Addison, Bennett; Staff, Department of Chemistry and Biochemistry, San Diego State University
Holland, Gregory; Faculty, Department of Chemistry and Biochemistry, San Diego State University
Abstract: Spiders produce up to seven unique types of silk boasting a wide range of impressive mechanical properties, and as such spider silks are an attractive target for biomimetic materials. For example, egg case (tubuliform) fibers display properties that rival high-tensile steel and Kevlar (high tensile strength and low extensibility) while, aciniform (prey-wrapping) silks show moderate strength but elastin-like extensibility, resulting in extremely high toughness. Our research focuses on understanding the molecular protein structures that give rise to the mechanical properties of spider silks. More specifically, we utilize mechanical testing and nuclear magnetic resonance (NMR) spectroscopy to investigate the role that protein secondary structures play in the various mechanical properties of spider silk fibers. Previous research suggests that polyalanine, or Poly(A)-rich -sheet nanocrystalline domains within spider silks are responsible for high strength, while -helical, unstructured and turn-like structures contribute to fiber extensibility, including the prey wrapping silks. However, direct links between the primary protein sequence and resulting mechanical properties have not been characterized adequately. Here we attempt to identify and quantify the relationships between the primary protein sequences, protein secondary structures, and resulting mechanical properties. Furthermore, the interaction with water has been shown to induce structural, dynamic and mechanical changes in silk fibers. For example, dragline silks are known to undergo supercontraction a process where the silk absorbs water, swells in diameter and shrinks in length. In our lab, we have observed that prey wrapping silk undergoes a change in dominant secondary structure from -helical to -sheet during the fiber wetting/drying process but, the underlying molecular mechanisms responsible for this remain unclear. We combine our mechanical data measurements with solid-state NMR to understand the molecular structural and dynamic variations that give rise to the observed mechanical properties on both dry and wet fibers. On this basis, we propose that the structural changes observed by NMR can be used to rationalize mechanical variation in web-construction, prey wrapping and egg case silks. Spider silks therefore, provide a dynamic range of tunable properties from which the future of material science can learn valuable lessons.
Poster #: 171
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Biochemistry, Nanoscience, Lipoproteins
Project Title: Transport of Chemotherapeutic Agent Paclitaxel by Apolipoprotein E3 containing Nanodiscs
Author List:
Benedicto, Vernon; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Ly, Brendan; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Narayanaswami, Vasanthy; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract: High-density lipoprotein (HDL) is a large spherical protein-lipid complex that plays a role in transport and delivery of lipids in the plasma. It is composed of proteins such as apolipoprotein E3 (apoE3), which have the ability to bind lipoprotein receptors that facilitate cellular uptake of lipoproteins. We propose to use this feature of apoE3 to incorporate hydrophobic chemotherapeutic agents such as paclitaxel (pac) into HDL for eventual uptake into cells. We will test the overall hypothesis that pac delivered via apoE3-containing reconstituted HDL (rHDL nanodiscs) is more efficient and specific in cellular uptake in glioblastoma cells than that delivered in currently used treatments as liposome formulations. The objectives of the current study are to: (i) isolate and purify recombinant human apoE3 bearing the lipoprotein receptor binding N-terminal (NT) domain (residues 1-191) with a His-tag; (ii) prepare rHDL using synthetic phospholipids and apoE3 NT domain; and, (iii) incorporate pac into rHDL and characterize pac loaded-rHDL. Overexpression of apoE3 NT domain was accomplished using E. coli cells and the protein purified by affinity chromatography. The protein was reconstituted with 1,2-dimyristoyl-sn-glycero-3-phosphocholine by sonication method in the absence or the presence of a mixture of pac and fluorescein-labeled pac analog (f-pac) to form rHDL nanodiscs. The rHDL nanodiscs were isolated by density gradient ultracentrifugation followed by fractionation. In a separate approach, pac/f-pac mixtures were directly added to preformed rHDL nanodiscs. The fractions were characterized in terms of protein, lipid and f-pac composition. Fluorescence emission spectra of f-pac/rHDL fractions revealed a significantly blue-shifted emission peak compared to emission of f-pac in DMSO. This finding suggests that f-pac is located in a highly hydrophobic environment such as that expected in the core of HDL and that pac was incorporated successfully into rHDL to yield pac/rHDL. Further studies will be pursued to determine the effect of pac/rHDL on microtubule formation in glioblastoma cells in comparison with that delivered in liposome formulations. Successful intracellular delivery of pac/rHDL offers a specific mode of entry of chemotherapeutic agents into cancer cells, which have high expression levels of lipoprotein receptors.
Poster #: 172
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Endoplasmic Reticulum stress, Unfolded Protein response, Activating transcription factor 6
Project Title: Effect of Girdin Depletion on Activating Transcription Factor 6 Processing During Endoplasmic Reticulum Stress
Author List:
Nguyen, Peter; Graduate, Chemistry and Biochemistry, California State University, Long Beach
Husain, Aida ; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Villegas, Reyalyn; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Bhandari, Deepali; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract: The endoplasmic reticulum (ER) is an organelle that plays a role in many important cellular functions including post-translational modification and folding of secretory and membrane proteins. Various pathophysiological conditions that result in disruption of ER function such as cancer, neurodegeneration and type II diabetes can trigger ER stress. Upon facing ER stress, cells activate an evolutionarily conserved signaling program called the Unfolded Protein Response (UPR) that aids in re-establishing normal ER functions. Our laboratory has recently identified that Girdin aka Gα-Interacting vesicle associated protein (GIV) plays a role in promoting cell survival during ER stress. This project focused on the role of GIV in the UPR, specifically on one of the UPR sensor proteins called the Activating Transcription Factor 6 (ATF6). We used the cervical cancer cell line HeLa as our model system. Stable cell lines expressing either control or GIV specific shRNA were transfected with the ATF6 construct. The cells were then treated with dithiothreitol, which triggers stress by reducing disulfide bonds needed to properly fold proteins in the ER lumen. Our western blotting analysis showed that GIV depleted cells expressed ~2X higher level of ATF6 as compared to control cells. GIV depleted cells also showed a higher level of ATF6 processing resulting in the release of the active N-terminal fragment of ATF6 from the rest of the protein as determined by western blotting for the cleaved N-terminal fragment of ATF6. Our current and future goals include determining the molecular mechanism by which GIV negatively regulates ATF6 expression and processing during ER stress.
This work was supported by the NIGMS Grants #SC2GM121246 and UL1GM118979; and CSUPERB New Investigator Grant to DB. AH and RV were supported by the NIGMS Awards #R25GM071638 and RL5GM118978, respectively.
Poster #: 173
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Phosphorylation, Cyclin dependent kinase 5, cell migration/proliferation
Project Title: Identification and characterization of a novel phosphoregulatory site on cyclin-dependent kinase 5
Author List:
Roach, Brett; Graduate, Chemistry and Biochemistry, California State University, Long Beach
Ngo, Jordan; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author, Nagel Award Finalist
Limso, Clariss; Graduate, Chemistry and Biochemistry, California State University, Long Beach
Oloja, Koyinsola; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Bhandari, Deepali; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract: Reversible protein phosphorylation serves as a key mechanism to regulate virtually every cellular process by altering the activation status, subcellular location, stability and/or protein-protein interactions of the target protein. Protein kinases, the enzymes that catalyze the phosphorylation reaction, are often themselves subject to phosphoregulation. Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase essential for embryonic development whose over-activation has been implicated in several pathologies including neurodegeneration, cancer cell metastasis and type II diabetes. Therefore, it is important to investigate molecular mechanism(s) that mediate regulation of CDK5 activity. Here, we identify and characterize a novel phosphoregulatory site on CDK5. Our mass spectrometry analysis identified seven putative phosphorylation sites on CDK5. The structural analysis predicted that phosphorylation of one of the identified sites, S47, would render the kinase catalytically inactive which we confirmed experimentally using the phosphomimetic and non-phosphorylatable mutants. The inactivation of the kinase due to the phosphomimetic change at S47 results from the inhibition of its interaction with its cognate activator, p35 as determined by co-immunoprecipitation assays. Finally, we connected the effect of this regulatory event to a cellular phenotype by showing that the S47D CDK5 mutant inhibits cell migration and promotes cell proliferation using wound scratch assays and phospho-Histone H3 immunostaining, respectively. Together, these results have uncovered a potential physiological mechanism to regulate CDK5 activity. The evolutionary conservation of this residue in not only CDK5, but also in other CDK family members suggests that this phosphosite may represent a shared regulatory mechanism across the CDK family.
This work was supported by the CSULB Office of Sponsored and Research Programs grant to DB. DB is also supported by the NIGMS Grant #SC2GM121246. JN and KBO are supported by the NIGMS awards #T34GM008074 and #R25GM071638, respectively.
Poster #: 174
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: apolipoprotein, protein structure, helix bundle
Project Title: Can the helix bundle of apolipophorin III reform after cleavage into two fragments?
Author List:
Cuellar, Andres; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Horn, James; Graduate, Chemistry and Biochemistry, California State University, Long Beach
Weers, Paul; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract: Apolipophorin III (apoLp-III) is an 18 kDa apolipoprotein present in the hemolymph of insects. The protein is composed a bundle of 5 amphipathic α-helices and switches to an open conformation to facilitate transport of lipids when there is a physiologically need, such as during flight or starvation. Because of the availability of high resolution structures and similarity with its mammalian equivalents, it is used as a model for structure-function studies. Hydrophobic residues are buried in the protein interior, and a conformational change results in exposure of a large continuous hydrophobic surface provided by non-polar faces of the amphipathic α-helices, which contact the lipid surface. The opening of the helix bindle is reversible, and the current project aims to investigate the contribution of hydrophobic forces to reform the helix bundle when the protein switches back to the lipid-free form. To this end, the protein was digested into two fragments, and their recombination was studied by spectroscopy. A point mutation was introduced at Ser-94, located in the loop connecting helix 3 and 4. This serine residue was changed to methionine to facilitate cleavage with cyanogen bromide. The digested protein was separated by RP-HPLC, yielding an N-terminal (NT) fragment of 9.8 kDa (helices 1-3), and a C-terminal (CT) fragment of 7.5 kDa (helices 4-5), as determined by MALDI-TOF. Tryptophan fluorescence studies showed a change in λmax from 332 nm for uncleaved apoLp-III to 355 nm for the CT fragment, which contains the only two tryptophan residues in the protein. This indicated full exposure of tryptophan residues in the CT fragment. Far UV circular dichroism showed that digestion reduced the helical content from 67% for the uncleaved protein to 22% α-helix for the NT fragment and 15% of the CT fragment. To reform the helix bundle, CT and NT fragments were pooled using a 1 to 1 molar ratio, but tryptophan fluorescence and circular dichroism spectroscopy showed no changes in the spectral properties of the fragments. Thus, the two fragments did not re-assemble into a helical bundle. Since the experiments were carried out in dilute protein solutions (< 0.2 mg/mL), additional studies will be carried out with more concentrated protein solutions (up to 2 mg/mL). Further, the helix inducing solvent trifluoroethanol will be used to increase the helical content of the two fragments, as their recombination may require a higher helical content.
Poster #: 175
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: Transcription Factors, U6 snRNA Transcription, Drosophila melanogaster
Project Title: Mechanism of assembly of the Small Nuclear RNA Activating Protein Complex and TFIIIB on the U6 snRNA gene promoter in Drosophila melanogaster.
Author List:
Kim, Mun Kyoung; Postdoc, Chemistry and Biochemistry, San Diego State University, Presenting Author
Phan, Phuc; Graduate, Chemistry and Biochemistry, San Diego State University
Wolfe, Angela; Undergraduate, Chemistry and Biochemistry, San Diego State University
Stumph, William; Faculty, Chemistry and Biochemistry, San Diego State University
Abstract: RNA polymerase II transcribes the spliceosomal snRNAs U1, U2, U4, and U5, but U6 snRNA is transcribed by RNA polymerase III (Pol III). Transcription by these distinct RNA polymerases is governed by different yet similar regulatory promoter elements upstream of the snRNA genes. For U6 snRNA transcription by Pol III, the small nuclear RNA activating protein complex (SNAPc) and TFIIIB must bind to and assemble on two promoter elements: a proximal sequence element (PSE) and a TATA box, respectively. In Drosophila melanogaster, DmSNAPc consists of three subunits: DmSNAP190, DmSNAP50 and DmSNAP43; likewise, TFIIIB also consists of three subunits, most commonly TBP, Brf1 and Bdp1. In flies, Pol III utilizes the TBP-related factor 1 (TRF1) for transcription of tRNA and 5S RNA; interestingly, however, the canonical TBP is utilized for U6 snRNA transcription by Pol III.
Previously, using site-specific protein-DNA photo-cross-linking assays, we mapped the individual binding patterns of DmSNAPc and TFIIIB separately on the PSE and TATA box of the U6 snRNA gene promoter, respectively. Furthermore, electrophoretic mobility shift assays (EMSAs) demonstrated that DmSNAPc recruits Bdp1 to the U6 gene promoter in the absence of TBP and Brf1, and this complex can subsequently recruit TBP to form a more stable complex even in the absence of Brf1. With that in mind, we are further studying the assembly of DmSNAPc, Bdp1 and TBP on the U6 gene promoter by site-specific protein-DNA photo-cross-linking. When this complex was assembled, we found three novel binding patterns relative to the patterns observed with the individual proteins: 1) DmSNAP190 extends its binding further downstream of the PSE. 2) Bdp1 binds to the 3’ end of the PSE. 3) TBP cross-links to more positions within the TATA box. These findings indicate that each of the proteins makes additional contacts with the DNA when assembled together on the U6 promoter and likely explain the increased overall stability of the multi-protein/DNA complex.
In order to map Bdp1 binding to the U6 snRNA gene promoter, we are utilizing truncated Bdp1s in photo-cross-linking assays. We are also examining the binding patterns of these factors in the presence of Brf1. In the near future, we will use cross-linking mass spectrometry to investigate protein-protein interactions to further develop an architectural model of DmSNAPc and TFIIIB on the U6 gene promoter. (Supported by NSF and the California Metabolic Research Foundation.)
Poster #: 176
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Functional Selectivity, Computational Biophysics, Receptor Biochemistry
Project Title: Understanding G Protein Selectivity of Dopamine Receptors Using Computational Biophysical Methods
Author List:
Portillo, Jennifer; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Abrol, Ravinder; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract: Dopamine is important in physiological processes associated with the rewards and pleasure system. Its function is facilitated by five G Protein – Coupled Receptors (GPCRs). The coupling that occurs between the dopamine receptor and G protein dictates the signaling cascade. The dopamine receptors D1 and D5 couple to the Gαs protein while the dopamine receptors D2, D3, and D4 couple to the Gαi protein. Dysregulation of the dopamine receptors can lead to neuropsychiatric disorders such as schizophrenia. These disorders are difficult to treat due to the side-effects that transpire. Understanding the coupling that occurs between the dopamine receptors and the G proteins can further assist with the development of pharmaceutical drugs by helping to alleviate some of the side-effects. Using computational biophysical methods, the coupling between the dopamine receptors and G protein is being studied to understand the structural mechanisms observed in G protein selectivity of these receptors. The dopamine receptor structures for D1 and D2 in complex with the two G proteins (Gαs and Gαi) have been relaxed in the membrane environment. A structural and thermodynamic analysis of those complexes in combination with structural bioinformatics methods has elucidated receptor residues playing a role in the G protein coupling, which has led to the design of receptor mutants with altered G protein selectivities. These mutants are now being characterized by computational biophysical methods before they will be tested in biochemical assays.
Poster #: 177
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Drug solubility, enzyme inhibition, cholinesterases
Project Title: Investigating Cosolvent Effects on Cholinesterases to Aid in Drug Solubility
Author List:
Novales, Noelle ; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Schwans, Jason; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract: Identification of cosolvents that solubilize pharmaceuticals while having minimal effects on protein structure is a key component in the screening of new drugs. In addition, the effect of organic cosolvents on protein structure or activity is a powerful tool in biochemical studies. Nevertheless, predicting the effects of cosolvents on enzyme activity is complicated, as even enzymes with similar overall structures elicit different behaviors in different cosolvents. Indeed, for the two major classes of cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), although the overall structures are essentially superimposable, previous studies reported that at low DMSO concentrations, AChE activity was reduced, but BChE activity was not changed. Building on this observation, we hypothesized that BChE activity was less sensitive to organic cosolvents. Initial cosolvent screening experiments revealed that AChE activity was decreased under all conditions tested, but BChE activity not affected for several cosolvents. Further, BChE activity was unchanged in solutions containing up to 50% methanol or ethylene glycol. To evaluate the effects of the cosolvents on catalytic activity (kcat) and substrate binding (KM), Michaelis-Menten kinetics experiments were performed for multiple common organic cosolvents, e.g., methanol, ethylene glycol, DMSO, and acetonitrile. All cosolvents had a modest effect on kcat (three-fold and less than two-fold for AChE and BChE, respectively), but KM values increased up to 20-fold for AChE and 10-fold for BChE, suggesting the cosolvents may occupy the active site and affect substrate binding. Evaluating if the cosolvent effects were reversible showed that in ethylene glycol AChE and BChE activity was restored to initial levels upon dilution of the cosolvent, but in methanol only 10-50% activity was observed after dilution. Together, the data suggest that BChE demonstrates more robustness to its cosolvent environment compared to AChE, but not all cosolvent effects are reversible. The results may enable better prediction of what cosolvents can be used for drug solubility and may allow us to select more ideal conditions to evaluate the potency of inhibitors for therapeutic applications.
This research was supported by NIH Award Number R25GM071638 and RL5GM118978. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Poster #: 178
Campus: CSU Los Angeles
Poster Category: Biochemistry
Keywords: Drosophila, Mad, Phosphorylation
Project Title: Terminating Bone Morphogenetic Protein Signals at the level of Mad linker phosphorylations
Author List:
Poole, Neil; Graduate, Biological sciences, California State University, Los Angeles, Presenting Author
Muradyan, Volodia; Graduate, Biological sciences, California State University, Los Angeles, Presenting Author
Eivers, Edward Eivers; Faculty, Biological Sciences, California State University, Los Angeles
Abstract: Bone morphogenetic protein (BMP) signaling has been found to be critically involved in a multitude of biological processes from embryonic tissue specification at the beginning of an organism’s life, to maintenance of tissue homeostasis in the adult. The significance of this signaling cascade is most evident in the array of human diseases (such as cancer) associated with dysregulated BMP signaling. The goal of the Eivers’ lab is to investigate how BMP signals in rapidly dividing embryonic tissues are fine-tuned at the level of Mad linker phosphorylations (Mad is the transcription factor which transduces the BMP signal). Overall, the BMP pathway is highly conserved throughout the animal kingdom, even down to the specific Mad phosphorylation sites of interest, thus allowing us to utilize the genetically amenable and rapidly developing fruit fly (Drosophila melanogaster) as our experimental model organism. Previous work by us and others has shown that phosphorylation of Mad linker serines initiates a sequence of cellular events resulting in Mad degradation by the proteasome, thus terminating BMP signaling. Here we provide evidence that Mad linker phosphorylations may also be involved in regulating BMP receptor degradation, thus potentially adding a second layer of control to the termination of the BMP pathway. Using fluorescent microscopy to image Drosophila wing tissues at the cellular level, we found that linker phosphorylated Mad localized to sub-cellular punctate structures, which co-localized with endocytosed type 1 BMP receptors. Western blot analysis also showed that mutation of Mad linker serines into non-phosphorylatable alanines or inhibition of the proteasome were able to stabilize type 1 BMP receptors when compare to non-mutated forms of Mad or control situations. In conclusion, our experiments aim to expand our knowledge of the BMP pathway by advancing our understanding of the basic molecular mechanisms which act to control the BMP signal at the level of protein stability.
This work was supported by NIH grant R15GM123374
Poster #: 179
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: Gut-brain axis, transfer-RNA, X-ray crystallography
Project Title: Overexpression, Purification and Crystallization of the Queuine Salvage Protein DUF2419
Author List:
Zarghan, Kevin; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Bayooz, Susan; Staff, Chemistry and Biochemistry, San Diego State University
Luthra, Amit; Postdoc, Chemistry and Biochemistry, San Diego State University
Swairjo, Manal; Faculty, Chemistry and Biochemistry, San Diego State University
Abstract: Queuine is a modified nucleobase that is biosynthesized de novo in bacteria and archaea. It is up taken by eukaryotes where it acts as a precursor of the modified nucleoside Queuosine (Q), found at the wobble base of cytoplasmic and mitochondrial tRNAs that incorporate Tyr, Asp, Asn and His amino acids. In human, queuine is exclusively retrieved from ingested food and microflora and is subsequently inserted in human tRNA by the human tRNA-guanine transglycosylase enzyme. Queuine is the only known micronutrient that directly affects efficiency and accuracy of translation of human proteins, with roles in cancer and healthy aging of the brain. It is required for the biosynthesis of BH4, a vital cofactor for numerous enzymes in the body, including those involved in formation of the amino acid tyrosine, and the key neurotransmitters dopamine and serotonin. Recently, DUF2419 was identified as a protein family involved in queuine salvage in eukaryotes, and the same function was demonstrated for the human homolog C9orf64. C9orf64 is deleted in cases of Acute Myeloid Leukemia, its promoter region is methylated in breast cancer cells, and levels of Q-modified human tRNA correlate with tumor aggression. Although, the exact biochemical function of DUF2419 remains unknown, its predicted structural similarity to 8-oxoguanine-DNA glycosylase, which cleaves oxidized guanines in DNA, suggests a ribonucleos(t)ide hydrolase activity to liberate queuine from its nucleos(t)ide precursor. We are testing this hypothesis through a combination of biochemical and structural methods. Here we report overexpression, purification and crystallization of human and bacterial DUF2419, to study function and structure. Homo sapein and Streptococcus thermophilus DUF2419 were cloned in pET28b vector, overexpressed in E. coli and purified to homogeneity and solubility of 5 mg/mL and 11 mg/mL, respectively. Using high throughput, vapor-diffusion crystallization methods, single crystals 5-200 m in size were grown from a variety of polyethylene glycol and salt conditions. With collection of X-ray diffraction data underway, uncovering the structures of these proteins will enable structure-guided mutagenesis to inform elucidation of function.
Funding: NIGMS grant GM110588 to M.A.S.; California Metabolic Research Foundation; U.S. DOE grant DEAC02–76SF00515; NIGMS grant P41GM103393; SDSU; CSUPERB.
Poster #: 180
Campus: San Francisco State University
Poster Category: Bioengineering
Keywords: Skin properties, Skin biomechanics, Biomechanical properties
Project Title: Development of a in-vivo biomedical tool to detect changes on Human Skin
Author List:
MO, Ed; Undergraduate, Engineering, San Francisco State University, Presenting Author
Coto, Miguel; Undergraduate, Engineering, San Francisco State University, Presenting Author
Azadi, Mojtaba; Faculty, Engineering, San Francisco State University
Abstract: Obtaining the mechanical properties of the skin stratum corneum and epidermis in-vivo will provide medical professionals with a way to obtain and record quantitative values on their reports. The stratum corneum and epidermis are 20 and 130 µm thick, respectively. Many of the tools are generally much larger than this length scale, which consequently may result in the mechanical properties of undesirable layers below the skin. In this study, the instrument that is used the Atomic Force Microscopy (AFM), is capable of capturing mechanical properties of stationary soft materials at the microscale and is fundamentally and theoretically superior in terms of collecting accurate data from small and stationary samples. However, using AFM on live human subjects in microscale imposes experimental challenges. A stabilizing setup was developed to minimize the subject’s hand motion and results in reliable data collection from the AFM. A 5µm radius spherical probe with maximum indentation depth of 2µm is used to detect the skin elastic modulus of samples in various parts of the human arm, as well as fixed/dried skin samples in-vitro. Furthermore, the Force Curves obtained from indentation of the AFM were analyzed in accordance with the Hertz Model and the resulting stiffness value is compared with the data found in the literature. An elastic modulus of 36 ± 19.42 MPa (mean, SD) was the result obtained for in-vivo testing. Moreover, when testing 2 in-vitro samples of skin taken from different sites of the arm, a Young’s Modulus of 10.04 ± 1.891 MPa (mean, SD) and 3.810 ± 0.53 MPa (mean, SD) were recorded. We propose that the difference of the in-vitro results may be due to the absence of softer living cell layers on the in-vitro sample or the vast range of the sample as the collagenic fibers and elastin are proportionately different among on any particular locus. Future work will provide more information on subjects that possess different skin characteristics (i.e dryness, sensitivity, melanin count, and complexion) as well as skin conditions (i.e infections, allergies, cancer), this allows for the feasibility of a quantitative practical method for the use of clinical diagnosis on skin conditions as the current technique used by medical practitioners heavily relies only on qualitative observations.
Poster #: 181
Campus: Cal Poly San Luis Obispo
Poster Category: Bioengineering
Keywords: Arteriogenesis, Macrophages, Ischemia
Project Title: Quantification of Pericollateral Macrophages During Arteriogenesis in a Diet Induced Obese Mouse Model
Author List:
Landon, Mark ; Graduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Hubbard, Madison; Graduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Cardinal, Trevor; Faculty, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Abstract: Peripheral artery disease (PAD) is caused by atherosclerotic plaque accumulation, which results in loss of blood perfusion to downstream tissues. Collateral arterioles can serve as natural bypasses following feed artery occlusion and undergo arteriogenesis (outward remodeling) to normalize shear stress and accommodate excess blood flow. Macrophages are necessary for arteriogenesis and their quantity is positively correlated with the extent of arteriogenesis. Obesity is a common co-morbidity of PAD and is known to impair arteriogenesis. Understanding macrophage recruitment in obese animal models could explain why arteriogenesis is impaired in obese patients. In this study, peripheral occlusions were mimicked through surgical ligation and novel methodology was used to quantify and compare pericollateral macrophages in diet-induced obese (DIO) and healthy lean mice. Lean mice had 1.3 fold more pericollateral macrophages than DIO mice in tissue undergoing arteriogenesis. Bone marrow cell transplantation altered pericollateral macrophage quantities in DIO mice to be more similar to lean controls. While future studies are needed to assess the phenotypic differences in pericollateral macrophages of DIO and lean mice during arteriogenesis, this study sets the foundation in understanding quantitative differences in pericollateral macrophages during arteriogenesis in an obese mouse model.
Poster #: 182
Campus: San Francisco State University
Poster Category: Bioengineering
Keywords: Tissue rejection, bioimpedance, bioengineering
Project Title: Wireless Bioimpedance Meter for in-vivo Transplanted Tissue Monitoring
Author List:
Bray, Shawn; Graduate, Engineering, San Francisco State University, Presenting Author
Jiang, Hao; Faculty, Engineering, San Francisco State University, Presenting Author
Abstract: Recent studies have shown that there is a correlate between transplanted tissue rejection and its electrical impedance variation. Real-time impedance monitoring has the potential to become a low cost alternative compared to invasive and expensive tissue biopsies. To advance the in-vivo study of this technology, a small form-factor (1 inch by 1 inch), low-power, wireless impedance monitoring system was developed. A set of thermal-passivation titanium-dioxide coated tetrodes are employed to measure the tissue impedance of an organ. An op-amp based circuit is created to measure the bioimepdance at kilo-Hertz range using the four-point method. The measured voltage and current are further analyzed by AD5933, an impedance analyzing IC developed by Analog Device Inc., with 12-bit accuracy. The hardware system is able to measure the bioimepdance up to 250 KHz, while the most noticeable impedance change happens at 50 KHz. A low-power WiFi module is used to deliver the measured impedance information wirelessly to a PC or an IOS app. The whole device is powered by two 1.5V CR2450 button cell battery with the diameter of 1 inch. The whole system can last several hours without activating the power saving algorithm. The measured impedance has been validated by the measurement using a commercial network analyzer. The described device is one of the most critical devices that facilitate the future animal study to validate the technology of tissue rejection in transplant organs.
Poster #: 183
Campus: San Diego State University
Poster Category: Bioengineering
Keywords: Intraventricular Flow, Heart Failure, Mock circulatory loop
Project Title: Evaluation of an artificial pulse for Left Ventricular Assist Devices
Author List:
Ortiz, Sean; Undergraduate, Mechanical Engineering, San Diego State University, Presenting Author
Marquez-Maya, Nikolas; Undergraduate, Mechanical Engineering, San Diego State University, Presenting Author
Montes, Ricardo; Graduate, Mechanical Engineering, San Diego State University
Vu, Vi ; Graduate, Mechanical Engineering, San Diego State University
May-Newman, Karen ; Faculty, Mechanical Engineering, San Diego State University
Abstract: Left ventricular assist devices (LVADs) are mechanical pumps that are surgically connected to the left ventricle and aorta to treat heart failure. Intraventricular blood flow in LVAD patients is redirected to exit through the LVAD rather than through the aortic valve. The alterations to the fluid dynamics are associated with a high rate of thromboembolism and stroke. In an effort to reduce these complications, rotary flow LVAD (Abbott Labs, IL) has introduced an artificial pulse that is intended to improve the LV vortex dynamics and washout. The goal of this study was to measure the flow field of the LVAD-supported LV in a mock circulatory loop and evaluate the vortex dynamics of the artificial pulse. Experimental studies were performed with a silicone model of the LV with dilated cardiomyopathy and a rotary flow LVAD. Neutrally buoyant fluorescent particles were added to a viscosity-matched blood analog solution. A LaVision PIV system captured a 40 Hz ensemble-averaged image sequence of the 2-D velocity field of the LV midplane for the cardiac cycle. A range of LVAD speeds were tested from 4.8 to 6.4krpm, with the artificial pulse produced every 2s. Aortic pressure, aortic flow and LVAD flow were measured continuously during the study. Vortex structures were computed from the 2-D velocity field data by calculating the vorticity and using the Q criterion to identify the vortex boundaries. Vortex circulation, kinetic energy (KE), size, shape, position, and blood residence time were calculated during the cardiac cycle. LVAD and aortic flow increased from 2.27 L/min at 4.8krpm to 3.82 L/min at 6.4krpm, and the pressure pulse is clearly visible in the hemodynamics signal. The average vortex circulation is reduced compared to the normal cardiac vortex, but at the highest LVAD speed the CW vortex achieves a circulation of 77%, and the CCW vortex reaches 41% of the Pre-LVAD value. The KE reached only 30% and 22% for the CW and CCW vortices, respectively, at the same LVAD speed. Although the vortex circulation and KE is relatively small, the fluid movement that the artificial pulse generates, in combination with the high flow enabled by the LVAD, reduces blood residence time by 65% compared to the Pre-LVAD condition. The reduction in residence time is clinically associated with lower thromboembolic risk. The artificial pulse of the LVAD improves vortex dynamics as a function of LVAD speed, and improves blood residence time.
Poster #: 184
Campus: Cal Poly San Luis Obispo
Poster Category: Bioengineering
Keywords: bone, tissue engineering, gas foaming
Project Title: Gas Foamed Poly Lactic-co-Glycolic Acid Scaffolds for In Vitro Bone Growth
Author List:
Torres, Leah; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Thomas, Matthew; Graduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Vaché, Conor; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Anastos, Thèo; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Heylman, Christopher; Faculty, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Abstract: Tissue engineering offers a relatively new approach to repair or regenerate bone. Gas foaming combined with particulate leaching is a novel method for creating porous scaffolds with material properties desirable for engineering trabecular bone. The goal of this project is to investigate the viability of the gas foaming method for fabricating Poly (Lactic-co-Glycolic Acid) (PLGA) scaffolds and determining whether osteoblasts enhance scaffold material stiffness. Gas foaming processes were optimized and a single scaffold consisted of 100mg of a mixture of 80% salt and 20% PLGA (85 lactide: 15 glycolide) particles that were between 63 and 250μm in diameter. Scaffolds had a diameter of 5mm and an average height of 3mm. Scaffolds received one of four treatments: seeded with 1 million osteoblasts (n = 7) and cultured in Human Osteoblast Differentiation Medium (ODM), cultured in ODM without cells (n = 7), cultured in Dulbecco’s Phosphate Buffered Saline (DPBS) without cells (n = 7), or cultured dry (n = 4). All conditions were cultured on the scaffolds for 6 days in a humidified incubator at 5% CO2. While optimizing the gas foaming technique, scaffold porosity was evaluated using scanning electron microscopy. The optimized technique produced scaffolds with evenly distributed pores 30-40μm in diameter. Calcification of extracellular matrix, indicating osteoblast viability, was analyzed using Alizarin Red S staining of cryosectioned scaffolds after 6 days of culture (n = 3 scaffolds per treatment). Inverted optical microscope images of the sections were taken at 4x magnification and Alizarin Red S staining was quantified using ImageJ. Matrix calcification was confirmed by larger aggregates of Alizarin Red S-stained material in the scaffolds seeded with osteoblasts (mean: 49841 pixels) compared to those without osteoblasts (ODM mean: 7371 pixels and DPBS mean: 149 pixels). Finally, compression testing was used to evaluate material properties of the scaffolds that had received each of the four treatments (n = 4 scaffolds per treatment). While there was no statistically significant difference in elastic modulus between any of the treatments, all scaffolds were within the normal range of trabecular bone, indicating that the scaffolds alone were a suitable mechanical match for trabecular bone.
Poster #: 185
Campus: Cal Poly San Luis Obispo
Poster Category: Bioengineering
Keywords: Dual extrusion 3D printing, Rapid prototyping, Microfluidic devices
Project Title: Dual Extrusion-Based 3D Printing: A Rapid Prototyping Alternative for Electronic Microfluidic Device Fabrication.
Author List:
Jangid, Aditya; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author, Nagel Award Nominee
Strong, Brandon; Graduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Prabhu, Sid; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Martinez, Andres; Faculty, Biological Sciences, California Polytechnic State University, San Luis Obispo
Liu, Bo; Faculty, BioResource and Agricultural Engineering, California Polytechnic State University, San Luis Obispo
Martinez, Nathaniel; Faculty, Biological Sciences, California Polytechnic State University, San Luis Obispo
Abstract: Objective: To develop a method for rapidly prototyping electronic and capillary microfluidic devices via extrusion-based 3D printing.
Background: Current microfabrication techniques for microfluidic devices typically require complex, labor-intensive processes (i.e., photolithography, soft lithography, metal evaporation & sputtering), and often necessitate the use of clean rooms. An alternative method of economical and rapid prototyping is 3D printing. While there are many examples of 3D printing being used as a prototyping modality in biotechnology (i.e., prosthetics, biomedical implants), it has more recently been applied to the field of microfluidics. Since the current generation of microfluidics devices require the integration of electronic components, we utilized dual extrusion-based 3D printing, thereby allowing for the prototyping of multi-material microfluidic devices with integrated electronics. To our knowledge, there is currently no easy method of rapidly prototyping electronic microfluidic devices.
Experimental: Devices were designed in SolidWorks (modeling software), exported to BCN3D Cura (slicing program), and printed via BCN3D (dual-extrusion 3D printer). For devices with electronic components, conductive polylactic-acid (PLA) was inlaid within a non-conductive PLA framework to create an internal circuitry. Capillary devices were produced similarly, and subsequently analyzed via light microscopy imaging for channel measurements.
Results & Discussion: Multi-layered, multi-electrode devices were fabricated using this method. Statistical analysis showed there was no significant difference in functionality between printed electronic components across three generations of simple microfluidic devices (p>0.05, n = 21). Fully-functional capillary devices were also fabricated containing channels as small as 50 µm in width, however reproducibility of sub-75 µm was variable. Channels as small as 100 µm were readily reproduced. Rapid prototyping via dual extrusion-based 3D printing may ultimately allow for an easy, cost-effective, high-throughput, high-resolution, and single-step method of rapidly prototyping electronic microfluidic devices, as well as show similar functional reproducibility of devices.
Acknowledgements: Many thanks to CSUPERB, NSF Grant no. DMR 1709740, and Cal Poly Baker/Koob Endowment for supporting this work.
Poster #: 186
Campus: CSU Northridge
Poster Category: Bioengineering
Keywords: 3D Bioprinting, Drop-on-Demand Inkjetting, Bioink hydrogel
Project Title: Effects of DOD Piezoelectric 3D Bioprinting on the Printability of Hydrogels
Author List:
Meza, Ryan; Undergraduate, Mechanical Engineering, California State University, Northridge, Presenting Author
Li, Bingbing; Faculty, Manufacturing Systems Engineering and Management, California State University, Northridge
Abstract: The growing number of people in need of organ transplants has increased steadily over the past few decades; however, the number of organ donors has not seen a large increase in about 10 years. This shortage of biocompatible, viable organs has lead to an increasingly large number of organ-failure related deaths. As organ donor waitlists continue to increase to an all-time high, 3D Bioprinting has emerged as a promising technological advancement to possibly solve this problem. Tissue engineering through freeform fabrication has provided an opportunity to have an unlimited supply of biocompatible tissues and organs using one’s own cells. This opportunity has led to the development of various 3D Bioprinters, each requiring specific bioinks capable of securely encapsulating cells. In this study, the compatibility of various bioinks were examined using a Drop-on-Demand, piezoelectric inkjet 3D bioprinter. We tested the properties of various bioinks in relation to this device to determine optimal bioink composition and printing parameters.
To test various bioinks under extreme conditions, hydrogels were extruded on a MicroFab PH-46 (MicroFab, Plano, TX) micro dispensing subsystem. This system consists of a MicroFab Jet Driver responsible for controlling the DOD pulse, a MicroFab pneumatic controller used to adjust the back pressure, an 80um piezoelectric micronozzle used to extrude bioinks, and an imaging camera STC-MB33USB (SENTech America, Carrollton, TX) to observe the droplet formation process. Biomaterials used in this experiment include sodium alginate (0.25% w/v, 0.5% w/v, 0.75% w/v, and 1% w/v) and sodium alginate (0.5% w/v) with gelatin (0.1% w/v).
Various hydrogel concentrations and compositions, corresponding to various viscosities, correlate to different levels of extrudability. These hydrogels were extruded and tested against different printing parameters to determine the bioinks printability. Voltage, rise/fall time, dwell/echo time and frequency have a significant effect on the printability and formability of microspheres, which are used to encapsulate cells during the printing process. The purpose of this experiment is to alter and measure the relation of these parameters to the size of the microspheres, bioink used, distance traveled, and droplet printability. Overall, sodium alginate based bioinks have proven to be an adaptable material with a broad range of capabilities that maintained good printability despite altering parameters.
Poster #: 187
Campus: Cal Poly Pomona
Poster Category: Bioengineering
Keywords: titanium, hip implants, electrochemical tests
Project Title: A new class of titanium alloys for structural implants
Author List:
Bae, Jaewan; Undergraduate, Chemical and Materials Engineering, California State Polytechnic University, Pomona, Presenting Author
Pacheco, Carlos; Undergraduate, Chemical and Materials Engineering, California State Polytechnic University, Pomona, Presenting Author
Gomez, Vanessa; Undergraduate, Chemical and Materials Engineering, California State Polytechnic University, Pomona
Patel, Neelam; Undergraduate, Chemical and Materials Engineering, California State Polytechnic University, Pomona
Svidensky, Michael; Undergraduate, Chemical and Materials Engineering, California State Polytechnic University, Pomona
Ravi, Vilupanur; Faculty, Chemical and Materials Engineering, California State Polytechnic University, Pomona
Abstract: The need for longevity of hip implants is increasing as the life expectancy of the world’s population increases. Titanium and titanium alloys, in particular Ti-6Al-4V (wt%) (Ti64), are commonly used in these applications due to their favorable strength-to-weight ratio, biocompatibility and corrosion resistance. There are several issues with the long-term use of this alloy. One of these is the phenomenon of stress shielding in which the elastic modulus mismatch between the implant and bone leads to bone loss and subsequent loosening of the implant. Another is the release of metal cations into the body with potential deleterious effects such as neurological disorders. In addition, inflammatory responses can be triggered through the phenomenon of aseptic loosening in which an imbalance between the osteoclasts and osteoblasts leads to bone resorption. A new class of beta-phase titanium alloys (TNZ alloys), offers promising characteristics for orthopedic applications due to their lower elastic moduli (thereby minimizing stress shielding) and non-toxic characteristics. In this study, the corrosion behavior of the two TNZ alloys, i.e., Ti-39Nb-6Zr (wt%) and Ti-28Nb-20Zr (wt%) and Ti64 control coupons, was investigated through electrochemical testing. The testing was carried in accordance with the ASTM-G59 and ASTM-F2129 standards in phosphate buffered saline solution at normal body conditions (pH 7.4 ±0.2 at 37 ±1 °C) to investigate the corrosion behavior of two TNZ alloys, i.e., Ti-39Nb-6Zr (wt%) and Ti-28Nb-20Zr (wt%), which were compared to TNZ and Ti64 as baselines. The TNZ alloys displayed a higher corrosion potential (> -0.5 V vs. SCE) and lower corrosion current density (< 75 nA/cm2) as compared to the Ti64 control alloy. These results indicate that the TNZ alloys will be more stable in physiological environments and have the potential to last longer in the body. Therefore, the TNZ alloys are promising candidate alloys for biomedical devices in accordance to ASTM F2129-17.
The authors would like to gratefully acknowledge Retech Systems LLC and Kittyhawk Products for helping with the alloys and HIPing. Financial support from the LA section of NACE International, Western States Corrosion Seminar and the Western Area is gratefully acknowledged. The use of the SEM/EDS was made possible through an NSF MRI (DMR – 1429674) grant.
Poster #: 188
Campus: Cal Poly San Luis Obispo
Poster Category: Bioengineering
Keywords: bioconjugation, biomaterials, copper-catalyzed azide-alkyne cycloaddition reaction
Project Title: Using “Click” Chemistry for the Development of Novel Bioactive Materials
Author List:
Le, Angeline; Undergraduate, Chem/Biochem, California Polytechnic State University, San Luis Obispo, Presenting Author
Zweng, Juliana; Undergraduate, Chem/Biochem, California Polytechnic State University, San Luis Obispo, Presenting Author
Hunter, McKenzie; Undergraduate, Chem/Biochem, California Polytechnic State University, San Luis Obispo
Knox, Karly; Undergraduate, Chem/biochem, California Polytechnic State University, San Luis Obispo
Costanzo, Philip; Faculty, Chem/Biochem, California Polytechnic State University, San Luis Obispo
Oza, Javin; Faculty, C, California Polytechnic State University, San Luis Obispo
Abstract: There have been notable advancements in the field of materials chemistry toward the development of materials that are compatible with, and augmented by, biological systems for broad applications such as surgical implants, regenerative medicine, and antimicrobial surfaces. However, the ability to conjugate materials to biomolecules is restricted due to the limited toolbox of protein chemistry. The 20 natural amino acids are sufficient to sustain life on earth, but limited in the diversity of biological chemistries available for interfacing with materials chemistry. To overcome this limitation, we use an expanded genetic code to produce green fluorescence protein (GFP) comprised of 21 amino acids. The 21st non-canonical amino acid, p-azidophenylalanine, is functionally distinct to the natural protein chemistry and can be covalently linked to polymers containing alkyne functionalities. This copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) is well characterized for polymers, but only recently applied to protein-polymer conjugation. This form of “Click” chemistry results in a covalent bond between the azide and alkyne groups, enabling the biofunctionalization of polymers. Although the CuAAC reaction between GFP and polyethylene glycol (PEG) has been established in previous literature, we found it necessary to optimize this reaction as various literatures report conflicting experimental conditions and poor reaction yields. We report our efforts to optimize the CuAAC reaction between GFP and PEG in free solution by varying the reagent stoichiometries under differing conditions, such as reaction temperature and time duration. Successful bioconjugation is quantified using gel electrophoresis to observe the migration of GFP-PEG products versus free GFP reactants resulting. Our efforts have resulted in increased reaction yields compared to previously published conditions from 10% to over 50%. With our understanding of optimal reaction conditions and this method of validation, we plan to 1) adapt our methods to conjugate proteins onto solid surfaces, and 2) explore other proteins and polymer combinations with unique and useful materials properties. When combined, these efforts will pave the way for the development of novel bioactive materials. This work is supported by the Research, Scholarly, and Creative Activities grant program at Cal Poly, SLO.
Poster #: 189
Campus: CSU Northridge
Poster Category: Bioengineering
Keywords: Bone Implant, topology optimization, selective laser melting 3D Metal printing
Project Title: Topological design and additive manufacturing of porous metals for bone implant
Author List:
Sotelo, Christian; Undergraduate, Department of Mechanical Engineering, California State University, Northridge, Presenting Author
Olmedo, Jose; Undergraduate, Department of Manufacturing Systems Engineering and Management, California State University, Northridge, Presenting Author
Moldthan, Matthew; Undergraduate, Department of Manufacturing Systems Engineering and Management, California State University, Northridge
Li, Bingbing; Faculty, Department of Manufacturing Systems Engineering and Management, California State University, Northridge
Abstract: One of the critical issues in orthopaedic regenerative medicine is the design of bone scaffolds and implants that replicate the biomechanical properties of the host bones. Porous metals have found themselves to be suitable candidates for repairing or replacing the damaged bones since their stiffness and porosity can be adjusted on demands. The fabrication of porous metals has been extensively explored over decades, however only limited controls over the internal architecture can be achieved by the conventional processes. However, the advancements in computer aided modeling techniques and 3D printing technology have fostered the ability to provide unique implants that can be made to suit the anatomy of the patient for patient-specific implants. At the same time, topology optimization techniques have been developed to enable the internal architecture of porous metals to be designed to achieve specified mechanical properties at will. Thus implants designed via the topology optimization approach and produced by additive manufacturing are of great interest. This method is capable of producing bone implants on demand, is substantially beneficial to minimize production time, produce less waste material, and eventually provide faster patient treatment overall. A 3D CAD model has been generated through OsiriX MD from a patient 2D scanning data and optimized through topology optimization software solidThinking Inspire. This technological advancement has transformed the bone implant cluster by providing an improved alternative which incorporates bone fitting as compared to conventional compression plates. The Renishaw AM400 with the resolution of 20 microns, demonstrates the possibility of using selective laser melting (SLM) technology to fabricate bone implant with optimized lattice features for direct osseointegration while following pre-, peri-, post- processes. The prototype was printed using the Renishaw AM400 in Titanium Ti-6Al-4V clear resin at 40 micron accuracy. By using metallographic observation, surface coatings, and surface finish processes, it is capable of producing mechanical properties and surface finish specifications that adhere or surpass to current implant standards.
Poster #: 190
Campus: San Diego State University
Poster Category: Bioengineering
Keywords: heart failure, thrombus, fluid dynamics
Project Title: Quantative assessment of an experimental model using intraventricular fluid dynamics for the detection of LVAD pump thrombus
Author List:
Montes, Ricardo; Graduate, Mechanical Engineering, San Diego State University, Presenting Author
Salim, Saniya; Graduate, Mechanical Engineering, San Diego State University, Presenting Author
Vu, Vi; Graduate, Mechanical Engineering, San Diego State University
May-Newman, Karen; Faculty, Mechanical Engineering , San Diego State University
Abstract: Left ventricular assist device (LVAD) pump thrombus (PT) is a serious complication that often requires LVAD pump exchange surgery. When clots form in the LVAD, they often grow slowly over time, eventually occluding the LVAD inflow, a progression that is difficult to detect with current treatment standards. Oftentimes, thrombosed LVAD patients will present high hemolysis and LDH, but these are nonspecific markers. Most patients with thrombus will have difficulty with LVAD unloading, indicating a clot has formed. However, by that point, the thrombus is usually too large to treat with increased anticoagulant therapy and a pump exchange must be performed. Pump replacement can be avoided if thrombus formation is detected early and treated. Recently, the S/D velocity ratio of the LVAD inflow cannula (IC) has been identified as a sensitive index. Our goal in this study was to assess the S/D ratio and hemodynamics in the detection of PT using an experimental model with matched flow conditions in a mock circulatory loop.
Experimental studies were performed with a silicone model of the LV with dilated cardiomyopathy and the HeartMate II LVAD. A particle image velocimetry system captured an ensemble-averaged image sequence of a 2-D velocity field of the LV midplane. A range of LVAD speeds were tested from 8 – 11krpm at baseline and during PT. PT was simulated with small disks centered over the rotor inflow housing, reducing the orifice area between 17 – 76%. Hemodynamics were measured continuously at 200 Hz. Localized velocity was calculated for a region of interest (ROI) at the entrance of the IC, to obtain the ratio of systolic maximum (S) to diastolic minimum (D).
LVAD to total flow ratio decreased as PT increased, determining an increase in outflow through the aortic valve opening, an investigated representative of PT. Comparable to clinical data, the ROI analysis revealed D decreasing and S remaining constant at the LVAD IC with an increase of PT, producing an increase in the S/D ratio.
Overall, PT increased resistance to LVAD inflow that increases with LVAD speed. The hemodynamics revealed a higher amount of aortic valve opening as PT obstruction reached 40%. The difference in S/D ratio between baseline and PT suggested a ratio of less than 10% to be small thrombus, a 10 – 25% ratio to be medium thrombi, and any S/D ratio over 25% to be large PT.
Poster #: 191
Campus: CSU San Marcos
Poster Category: Bioengineering
Keywords: Stem Cells, Cytoskeleton, Numb
Project Title: Cell Fate Determinant Numb Localizes in the Cell Pseudopodia of Human Adipose-Derived Stem Cells
Author List:
Javier, Areli J. ; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Burch, Shalise; Graduate, Biological Sciences, California State University San Marcos, Presenting Author
Nguyen, Mary; Undergraduate, Biological Sciences, California State University San Marcos
Jacobo, Daniel; Undergraduate, Biological Sciences, California State University San Marcos
Luna Lopez, Carlos; Faculty, Biological Sciences, California State University San Marcos
Abstract: Maintaining stem cell self-renewal and promoting differentiation remain inefficient processes that hinder the effectiveness of stem cell therapies and bioengineering applications. These processes could be regulated by identifying the mechanisms responsible for the localization of fate determinant factors inside the cell and during cell division. For example, the asymmetric distribution of Numb has been shown to regulate differentiation during cell division.
The cell cytoskeleton network is responsible for the dynamics of many cell compounds, such as organelles, proteins and RNA. In this work we seek to understand if the interactions of the cell cytoskeleton and the mechanical substrate affect the localization of Numb proteins in human adipose-derived stem cells.
We used human adipose-derived stem cells (HADSCs) and created substrates of 1 Mpa and 0.1 MPa stiffness using polydimethylsiloxane (PDMS). We used 1GPa glass substrates for comparison. Substrates were coated with Poly-d-lysine for 30-min to promote cell adhesion. We plated HADSCs and waited 24 hours before imaging. We stained cells with Numb primary antibody, 488 Phalloidin for the actin cytoskeleton and DAPI for the nuclei. We used a Keyence Fluorescence Microscope to image 50 cells from 6 independent experiments. We analyzed cell area and total cell fluorescence using ImageJ (NIH). For statistics between two sets of data we used an unpaired t-test with Welch’s correction.
We found that the total fluorescence expression of Numb in cells did not vary between PDMS and glass substrates (p=0.97). We found cells undergoing division, those with asymmetric expression of Numb tended to have a difference of 50% in fluorescence (p=.009). We did not find a difference in the number of cells with asymmetric expression of Numb between PDMS and glass substrates (p=0.34). Interestingly, we found that Numb was highly localized in the pseudopodia of spindle-shaped cells, independently of the substrate.
Our results indicate that Numb can be asymmetrically localized in HADSCs undergoing division, but the asymmetric division was independent of substrate stiffness. Instead, we found that Numb localization was found in cell pseudopodia and elongated cells. In the future, we will investigate the mechanism that induces Numb localization in pseudopodia and whether that mechanism can be used to regulate asymmetric cell division.
Poster #: 192
Campus: CSU Long Beach
Poster Category: Bioengineering
Keywords: Tissue Engineering, Muscle Repair, Collagen
Project Title: Bioengineered Tissue for Muscle Repair
Author List:
Padron, Kimberly; Undergraduate, Chemistry Department, California State University, Long Beach, Presenting Author
Nguyen, Jeffrey; Undergraduate, Biomedical Engineering Department, California State University, Long Beach, Presenting Author
Vu, Bryan; Undergraduate, Biomedical Engineering Department, California State University, Long Beach
Anderson-Fears, Keenan; Staff, Biomedical Engineering Department, California State University, Long Beach
Ayala, Perla; Faculty, Biomedical Engineering Department, California State University, Long Beach
Abstract: Severe tissue damage, due to injury or disease can result in scar formation and loss of tissue function. The objective of this research is to uncover novel strategies to reduce pathological fibrosis and promote growth of muscle satellite cells at the site of injury. Our approach relies on discerning how the interplay of cellular systems and extracellular matrix components and signals can be modulated by biochemical and physical cues. In this work, the effect of extracellular matrix (ECM) and micro-scale topographical factors on proliferation and gene expression is investigated in a bioengineered tissue scaffold. To fabricate the scaffold, collagen type 1 was extracted from porcine dermal tissue through acid solubilization. After purification, the collagen was then used to make a film that was casted onto a poly-dimethyl siloxane (PDMS) mold with micro channels. The frame for the PDMS well was designed in SolidWorks and 3D printed using a Project 6000 3D Printer. To assess cell proliferation and organization, C2C12 myoblasts were cultured in these substrates and were imaged using fluorescent microscopy at various times in order to capture their growth. The bioengineered tissue demonstrated formation of myofibrils and directed alignment of myoblasts within the channels. Results from this work will help to create future engineered treatments that promote muscle repair.
ACKNOWLEDGEMENTS:
This research was supported by CSUPERB, CSULB ORSP, CSULB COE SFG, CSULB UROP, and BUILD (National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers; 5UL1GM118979; 5TL4GM118980; 5RL5GM118978). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Poster #: 193
Campus: CSU Northridge
Poster Category: Bioengineering
Keywords: Ultrasound, Visualization, Wave scattering
Project Title: Visualization of the scattering of high-intensity focused ultrasonic waves at bone phantoms
Author List:
Brown, Matthew; Undergraduate, Mechanical Engineering, California State University, Northridge, Presenting Author, Nagel Award Finalist
Schaal, Christoph; Faculty, Mechanical Engineering, California State University, Northridge
Abstract: While high-intensity focused ultrasound (HIFU) is already being used for the ablation of tissue, such as in the case of prostate cancer, targeting tissue deeper inside the body remains challenging due to the effects of wave scattering and obstruction at bones. In this work, the effects of bones on the transmission of ultrasound in the body is investigated.
In laboratory experiments, the shadowgraph method is used to visualize the partial and complete obstruction of the ultrasonic beam path from a HIFU transducer operating at 670kHz by bone phantoms in water. The material of the bone phantoms is a fiber-filled epoxy composite, which mimics the properties of cortical bone. The used shadowgraph system uses a short pulsed laser that is synced with a CMOS camera. Since the ultrasonic waves cause a change in density and index of refraction, a bright and dark pattern, showing wave peaks and troughs, will appear in the images, thus visualizing the waves’ propagation, reflection and transmission. Ultrasonic wave transmission and reflection is captured, and the effect of obstruction on ultrasound focal point intensities is quantified. Images with no obstruction, partial obstruction, and complete obstruction are captured. In addition to visualization, preliminary measurements of changes in pressure at the focal point of the transducer due to the obstruction by bones are recorded using a hydrophone and compared to results obtained from the shadowgraph images.
The results of the experiments indicate that for certain obstruction cases, waves scatter in such a way that they destructively interfere with incident waves, leading to a decrease in focal point pressure. Furthermore, it is shown that the most significant reduction in focal point intensity occurs not for full but for partial obstruction due to the disruption in the shape of the focusing waves. Overall, this experimental work provides a baseline for future research in medical ultrasound. Most significantly, it can be used as a reference for the optimization of targeting specific tissue inside the body to which the direct path is obstructed by bone tissue, and thus leading to the expansion of HIFU for other types of cancers as well as other ailments.
Poster #: 194
Campus: San Diego State University
Poster Category: Clinical
Keywords: polycystic ovary syndrome, letrozole, organizational
Project Title: Determining the organizational and activational effects of hyperandrogenism in a letrozole-induced mouse model of polycystic ovary syndrome
Author List:
Arroyo, Pablo; Graduate, Biology, San Diego State University, Presenting Author
Thackray, Varykina; Faculty, Dept. of Reproductive Medicine, University of California, San Diego
Ho, Bryan S.; Graduate, Biology, San Diego State University
Sau, Lillian; Undergraduate, Biology, University of California, San Diego
Kelley, Scott T; Faculty, Biology, San Diego State University
Abstract: Polycystic ovary syndrome (PCOS) is an endocrine disorder that affects 10-15% of reproductive-aged women. Women with PCOS are diagnosed by having two out of the following features: hyperandrogenism, oligo- or amenorrhea, and polycystic ovaries. In addition, women with PCOS also suffer from a higher incidence of infertility, miscarriage, and pregnancy complications, as well as an increased risk for type 2 and gestational diabetes, cardiovascular disease and non-alcoholic fatty liver disease. Currently the etiology of PCOS remains unknown, with speculation that early excess androgen may play a role. Established studies in Caucasian and Han Chinese women have also shown that the gut microbiome may play a causal role in PCOS. To study PCOS, animal models such as rodents are used as they provide an important insight in studying excess androgens and metabolic dysfunction. One mouse model, the letrozole-induced mouse model, relies on letrozole to induce all three PCOS features by blocking the ability of aromatase to convert androgens to estrogens. While this model recapitulates the majority of known PCOS symptoms during and after puberty, it is unknown whether the hyperandrogenism induced by letrozole causes permanent changes in these mice. This has important implications for understanding the degree to which PCOS may be reversible in adulthood. To determine whether the effects of letrozole treatment were organizational (permanent) or activational (temporary), we compared physiological parameters between letrozole and placebo mice prior to and after the letrozole pellets were surgically removed. During these two periods, metabolic effects were measured via an insulin tolerance test, hormone assays, and glucose measurements while reproductive effects were analyzed through vaginal smears and ovarian histology. From the results, we found that most metabolic and reproductive parameters for letrozole treated mice normalized to placebo levels, with the exception of insulin. We also collected fecal samples every week for future studies on the gut microbial composition between letrozole and placebo treated mice. These results suggest that effects induced by hyperandrogenism are activational rather than organizational. In summary, these results can help suggest that the effects of PCOS may be reversible in adulthood.
Poster #: 195
Campus: CSU Long Beach
Poster Category: Clinical
Keywords: fall risks, Dual-tasking intervention, Older adults
Project Title: Improving Balance and Gait Using Dual-Task Intervention in Community-Dwelling Older Adults
Author List:
Tuttle, Zachary; Graduate, Psychology, California State University, Long Beach, Presenting Author
Krishnan, Vennila; Faculty, Physical Therapy, California State University, Long Beach, Presenting Author
Cho, Young-Hee; Faculty, Psychology, California State University, Long Beach
White, Barbara; Faculty, Gerontology, California State University, Long Beach
Olfat, Mohamed; Faculty, Physical Therapy, California State University, Long Beach
Abstract: Functional impairments such as increased step variability while walking due to age-related decline is a known fall risk for community-dwelling older adults (Hollman, Kovash, Kubik, & Linbo, 2007). Interventions aimed at reducing fall risks among community-dwelling older adults are becoming increasingly important. Physical training has been shown to improve balance (Cadore, Rodriguez-Manas, Sinclair, & Izquierdo, 2013). Even though falls occur more frequently during dual-tasking (i.e., performing another task while walking) than just walking, the effectiveness of dual-task training on the reduction of fall risks have not been investigated systematically (Agmon, Belza, Nguyen, Logsdon, & Kelly, 2014). This study investigated the effectiveness of dual-task training (i.e., physical training while simultaneously performing a cognitive task) on a clinical measure of balance and on walking performance in community-dwelling older adults. Eleven older adults, aged 73.3 ± 7.80 years, participated in a group exercise class (conducted 2 times/week for 8 weeks) emphasizing a simultaneous training of physical exercise and cognitive performance. Outcome measures were clinical balance assessed with the Fullerton Advanced Balance scale (FAB; Schlenstedt et al., 2016) and gait parameters assessed with a Zeno walkway. Gait parameters were assessed twice — one while walking and the other while walking and performing a cognitive Stroop task simultaneously. Dual-task walking costs (i.e., the decline of walking performance while performing a cognitive task simultaneously) were computed as the percentage normalized difference in gait parameters between the walking only condition and the dual-tasking condition. The results showed that the dual-task training effectively improved the participant’s balance on the FAB score (from M = 30.5 before training to M = 35.5 after training, p <. 001). The training was also effective in improving gait such that after the intervention, dual-task walking costs were significantly reduced in three gait parameters of gait cycle time (from 5.9% to 1.71%, p = .002), velocity (17.8% to 11.5%, p = .002), and cadence (from 5.0% to 0.9%, p = .02). These results underscore the effectiveness of an intervention targeting both physical and cognitive components simultaneously on improving balance as well as gait parameters and thereby reducing fall risks in community-dwelling older adults.
Poster #: 196
Campus: CSU Long Beach
Poster Category: Clinical
Keywords: Addiction, Psychostimulants, Development
Project Title: Adolescent male rats exhibit adult-like methamphetamine sensitization in one trial paradigm, while female rats do not.
Author List:
Sortman, Bo; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Franco, Daniela; Graduate, Psychology, California State University, Long Beach
Nunez, Eric; Undergraduate, Psychology, California State University, Long Beach
Moreno-Barriga, Cynthia; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Iniguez, Sergio; University of Texas, El Paso
Zavala, Arturo; Faculty, Psychology, California State University, Long Beach
Abstract: Behavioral sensitization is an augmented behavioral response to psychostimulant drugs, such as methamphetamine (METH). Sensitization can be induced after only two drug injections. Interestingly, young rats will display sensitization independent of the environmental context that is associated with METH, while adult rats will only display a sensitized response in the same context. Surprisingly, METH sensitization has not been examined in adolescent rats. Thus, we investigated the ontogeny of METH context-dependent sensitization in adolescent rats. We hypothesized that adolescent male and female rats would exhibit adult-typical one-trial sensitization. Male and female adolescent rats were injected either with saline or METH (3.0 mg/kg, i.p.) paired in their home cage (home-paired) or a novel activity chamber (activity-paired). The next day, rats in each group were given an injection of METH (0, 0.1, or 0.3 mg/kg, i.p.) immediately before being placed in the activity chamber and their locomotor activity was assessed for 90 min. Male rats displayed context-dependent sensitization at any dose of METH examined, whereas females only showed enhanced locomotion compared controls. These findings indicate that adolescent male rats exhibit adult-typical METH sensitization (i.e., context-dependent sensitization), whereas females continue to exhibit juvenile-typical METH sensitization. Sensitization to drug effects is thought to play a key role in the development of addiction. Understanding the development of context-dependent sensitization is important because context-independent sensitization may underlie increased vulnerability to develop addictive behaviors, given sensitization is evident independent of the context the drug is taken. Here we demonstrate that unlike young rats, male adolescent rats exhibit context-dependent sensitization. Further studies will need to examine the ontogeny of this sensitized response to identify the heightened susceptibility to abuse drugs in juvenile populations.
Poster #: 197
Campus: CSU Sacramento
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: enediyne cyclization, density functional theory (DFT), anticancer
Project Title: Biradical Stability in Bergman Cyclization Products from Benzannelated Enediynes
Author List:
Waltonen, Alexander; Undergraduate, Chemistry, California State University, Sacramento, Presenting Author
Gherman, Benjamin; Faculty, Chemistry, California State University, Sacramento
Spence, John; Faculty, Chemistry, California State University, Sacramento
Abstract: Enediynes are organic compounds which undergo Bergman cyclization when exposed to light or heat to form biradical compounds. The resulting biradical products can abstract hydrogen atoms from an appropriate donor, including the sugar backbone of DNA leading to cleavage of the DNA strands. This cytotoxic behavior has led enediyne compounds to be considered for possible antitumor drugs. Angularly benzannelated enediynes can have improved Bergman cyclization reactivity as they lead to more stable products compared to linearly benzannelated enediynes due to increased aromaticity gain in the former upon cyclization. Previous research showed an interaction between the biradical and the nitrogen atom of angularly benzannelated enediynes with quinoxaline supporting groups, which does not occur in the case of linear benzannelation. In this research, the effect of a supporting group heteroatom on the stability of biradical products from angularly benzannelated enediynes was examined. Comparison of a naphthyl support group with a quinoxaline support group was initially conducted to test the nitrogen lone pair interaction with the biradical. The nitrogen was also changed to boron, which lacks a lone pair, and phosphorous, whose lone pair occupies a larger orbital than nitrogen. Using Density Functional Theory (DFT), the biradical stability with the various linearly and angularly benzannelated enediynes was compared by calculating the singlet-triplet splitting energies, aromatic stabilization energies (ASE), biradical stabilization energies (BSE), nucleus-independent chemical shifts (NICS) for the biradical containing rings, and magnetic susceptibilities. Using phosphorous as the heteroatom showed increased stability in the angular products compared to the other heteroatoms studied, suggesting a favorable interaction between the radicals and the diffuse lone pair from phosphorous. At the other end of the spectrum, nitrogen as the heteroatom led to the least stable biradical products, likely due to electrostatic repulsion between the radicals and the much more concentrated lone pair electron density from nitrogen.
Poster #: 198
Campus: San Diego State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Bioinformatics, Dengue virus and Zika virus, cleaved substrates
Project Title: A bioinformatics approach for Dengue and Zika virus substrate discovery
Author List:
Salami, Majid; Graduate, Biology, San Diego State University, Presenting Author
Barr, Nina; Graduate, Biology, San Diego State University
Wolkowicz, Roland; Faculty, Biology, San Diego State University
Abstract: Dengue (DenV) and Zika virus (ZIKV) are two of the most important human pathogens. Understanding how these viruses affect the host at the biological level is a key component in order to find new cures to eradicate infection. In such an attempt we have undertaken a bioinformatics approach aimed at finding potential substrates of the viral proteases within the human proteome. Viral proteases, considered one of the main targets for antivirals, cleave, in addition to viral proteins, proteins of the host. By revealing these substrates we hope to further elucidate how viral infection affects the host at the cellular level. For that purpose, we have undertaken a bioinformatics approach and created a set of search parameters. These search parameters take into account the sequences within the viral proteome known to be cleaved by the viral proteases. These include the boundaries between Capsid and non-structural protein (NS) 2A (CA/NS2A), NS2A/B, NS2B/3, NS3/4A, NS4A/B and NS4B/5, the sequences of which were obtained using reliable, peer reviewed, sequences published and openly available from the NCBI site. Our search inquiries take into account the degree of interchangeable amino-acids in each position, length of cleavage recognition site and relative position of cleavage within the site. We have performed preliminary searches for the main four serotypes of DenV and for ZIKV. These searches led to a long list of hits that we have compiled in tables for the individual search parameters; 808 hits for the four serotypes of DenV and 58 hits for the 2015 ZIKV. This preliminary list contains all hits including their isoforms. We are now in the process of defining singlets within these lists which should account for the unique protein sequences. For instance we found EIF4G1, a protein required for protein translation, already described in literature as being cleaved by ZIKV, validating our approach for the search of substrates. Importantly, we also found hits that have not been described, such as Myomesin, involved in the structure of filament cytoskeleton. Our lab has the unique opportunity to assess if putative substrates found in silico are indeed cleaved by the virus. This will be done utilizing the cellular platform developed to monitor cleavage. In summary, by linking our bioinformatics approach with biological assays will reveal the biological significance of our findings in an attempt to fight DenV and ZIKV infection.
Poster #: 199
Campus: CSU Sacramento
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: metalloenzyme, density functional theory (DFT), catalytic mechanism
Project Title: Computational study of the reactivity and chemoselectivity of non-heme iron halogenases
Author List:
Lindsay, Dylan; Undergraduate, Chemistry, California State University, Sacramento, Presenting Author
Gherman, Benjamin; Faculty, Chemistry, California State University, Sacramento
Abstract: Chemoselective halogenation of aliphatic compounds is an important step in the biosynthesis of many natural products. The non-heme iron halogenase metalloenzymes CytC3 and SyrB2, which catalyze the production of antitumor and biosurfactant compounds, respectively, are two naturally occurring enzymes that halogenate alkanes at room temperature, are regenerative, and do not require light like traditional halogenation. In both enzymes, it is an oxoiron(IV)-halide active site species which is responsible for the halogenation reactivity. The reaction occurs in two steps – hydrogen atom abstraction to give a hydroxoiron(III) species with formation of an organic radical, followed by abstraction of a halide ligand from the iron complex to give the halogenated product. Recently, the first biomimetic model for these enzymes’ active sites capable of reproducing their chemoselective halogenation reactivity was developed and formulated as [FeIV(O)(TQA)(Cl/Br)]+ (TQA= tris(quinolyl-2-methyl)amine) (J. Am. Chem. Soc., 2016, 138, 2484-2487). In this research, using density functional theory (DFT) calculations, the energetics of the halogenation reaction of substrates toluene and cyclohexane catalyzed by the TQA complex are determined. In particular, the activation energy for the hydrogen abstraction step is first computed; following this, the reaction energies for the halogenation step and a competing hydroxylation step are computed based upon both radical rebound and cage escape mechanisms. Results indicate that halogen identity in the TQA complex has little effect on the hydrogen abstraction step, whereas a significant difference in the rate for the halogenation step is seen with chlorine versus bromine as the halogen. The halogenation chemoselectivity is evidenced in the second reaction step as seen through the difference in activation and reaction energies versus the potential hydroxylation reaction. The results here provide an essential first stride toward the long-term goal of better understanding the chemistry occurring in non-heme iron halogenases and in designing new biomimetic complexes with improved reactivity for these metalloenzymes.
Acknowledgements: Funding for this project has been provided by California State University, Sacramento to D. J. L. through a Russell-Forkey Research Award.
Poster #: 200
Campus: CSU Fresno
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Mosquito repellent, DEET, Computational Modeling
Project Title: Computational modeling of DEET analogs to gain insight and develop potent insect repellents
Author List:
Morrelli, Derek; Graduate, Chemistry, California State University, Fresno, Presenting Author
Krishnan, Krish; Faculty, Chemistry, California State University, Fresno
Maitra, Santanu; Faculty, Chemistry, California State University, Fresno
Abstract: Mosquitoes are well known as carriers of numerous pathogens, such as the Zika virus, West Nile virus, dengue fever, and malaria. Although mosquitoes are a continued growing health risk, their eradication is a highly debated topic for the downstream ecological impacts. N,N-diethyl-m-toluamide (DEET) is the most widely used repellent against mosquitoes, an effective way to reduce disease transmission by mosquitoes. Despite its widely accepted use, DEET does have room for improvement based on reasons such as, (1) human toxicity, (2) limited time of application, (3) chemical reaction with plastic, and (4) reduced effectiveness over continued use. To explore the effectiveness of various DEET analogs, computational simulations can potentially aide the practical design of analogs to accurately tune mosquito repellency without sacrificing essential practical properties. Using a combination of molecular modeling and simulation software Audodock and YASARA we have examined 14 DEET analogs and their interactions with the mosquito odorant binding protein, AgamOBP1. Autodock uses the crystal structure of DEET bound AgamOBP1 and replaces DEET with a different molecular entity, calculating the new binding affinity and dissociation constant. The binding affinities and dissociation constants were compared between all 14 DEET analogs as well as DEET. Molecules with a higher binding affinity and lower dissociation constant are expected to have a stronger interaction with the odorant binding protein, therefore acting as a more effective repellent. The accuracy of these predictions of effectiveness is compared with biological assay results when they are completed. The research team believes that the computational modeling can potentially enhance the identification of a more potent mosquito repellent retaining other essential properties for application in the real world.
Poster #: 201
Campus: CSU Fullerton
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: mutations, heat-shock, cell survival
Project Title: Molecular evolution and functional diversification of the human BCL2-associated athanogene-1
Author List:
Nguyen, Peter; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Chavez, Christina; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Nikolaidis, Nikolas; Faculty, Biological Science, California State University, Fullerton
Abstract: Molecular chaperones, and, in particular, the 70-kDa heat shock proteins (Hsp70s) are key orchestrators of the stress response system. To perform their critical cellular functions, Hsp70s require the presence of specific co-chaperones, which include the nucleotide exchange factors containing the BCL2-associated athanogene (BAG) domain. Bag-1 is one of these proteins that partake in a wide range of cellular processes including apoptosis, cell growth, cell migration, cell survival, cell differentiation, transcriptional regulation, protein refolding and degradation, as well as tumorigenesis. However, the origin and evolution of Bag-1 proteins between and within species are mostly uncharacterized. To elucidate the evolution and understand how natural variation alters the way we respond to environmental stress and disease, we investigated the macro- and micro-evolution of Bag-1 using orthologous sequences and single nucleotide polymorphisms (SNPs). We first collected and analyzed several orthologous Bag-1 sequences across animals, plants, and fungi, mapped intron positions and phase, reconstructed phylogeny, and analyzed protein characteristics. These data indicated that Bag-1 originated before animals, plants, and fungi split, but almost all fungal species lost the gene. Furthermore, although Bag1’s structure has remained relatively conserved, kingdom-specific conserved differences exist at sites of known function, suggesting functional divergence within each kingdom. We then analyzed SNPs from the 1000 genomes database to determine the evolutionary patterns within humans. These analyses revealed that the SNP-density is unequally distributed within the Bag-1 gene and the ratio of non-synonymous/synonymous SNPs is significantly higher than 1 in the Bag domain region, which is an indication of positive selection. To further explore this notion, we performed an intracellular refolding assay, and differently from our expectation, we found that only one out of five SNPs tested alters the major co-chaperone properties of Bag-1. Collectively, these data suggest that although the co-chaperone functions of Bag-1 are highly conserved and can probably tolerate several radical mutations, Bag-1 has acquired specialized and potentially unexplored functions within the evolutionary process.
Poster #: 202
Campus: CSU Channel Islands
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: nucleoside analogues, antimicrobial resistant, In silico modeling
Project Title: In silico modeling studies and pharmacokinetics–pharmacodynamic computational screening of novel nucleoside analogues
Author List:
Pineda, Richard ; Undergraduate, Chemistry, California State University Channel Islands, Presenting Author
Nguyen, Michelle ; Undergraduate, Chemistry, California State University Channel Islands, Presenting Author
Awad, Ahmed; Faculty, Chemistry, California State University Channel Islands
Abstract: Antimicrobial resistant bacteria are becoming a major threat and problem towards health care and its consumers. The World health organization has issued paramount warnings stating that by 2050 all bacteria will be resistant to all current antibiotics, and close to a million people die worldwide each year due to bacterial infections. As medicine begins to advance, the rise in antibiotic resistance increases. Through the approach of pharmacokinetics and internal coordinate mechanics algorithm (ICM), the mechanism for the active site of Metallo-ß-Lactamase (MBL), a target enzyme in antibiotic resistant bacteria drug development, was explored to yield several promising non-toxic novel nucleoside analogues that demonstrate favorable zinc deactivating activity. By examining the LD50 factor and Lipinski’s rule of five along with programs such as ADMETSAR and SWISSADME, possible compounds viable for further testing were proposed. A list of 100 compounds and five controls were isolated through computational screening. Analysis of the data demonstrated three non toxic lead compounds, 37N, 38V, and 64I, for three Metallo-ß-Lactamase subclass B1 proteins; New Delhi, Imipenemase 1, and Verona integron-encoded 2. The hydrogen bonding acceptor interactions between the lead novel compound and the hydrophobic binding pocket, contributed to its computational success. Nitrogen and Oxygen on the bases chelated best with the active site. Compound 37N formed H-bonds between 8-N:Q123, 3-N:17-H and 27-O:D223. Compound 38V formed H-bonds between 8-N:D117, 9-O:N148, and 30-O:R205(2x). Compound 64I formed H-bonds between 7-O:Zn1, 9-O:Y163, and 27-H:G164. The pharmacokinetic and physiochemical profiles of these novel compounds were examined and isolated for their future potential viability.
Poster #: 203
Campus: CSU Fullerton
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Computational Chemistry, Organic Chemistry, Reactive Intermediates
Project Title: A Computational Approach Towards Developing a Green Synthesis Route for Organic Heterocyclic Scaffolds for Pharmaceuticals
Author List:
Gillette, Jonathan; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Abiad, Amanda; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Torres, Danielle ; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Petit, Andrew; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Abstract: Heterocycles are molecules containing at least one ring in which one or more atoms is not carbon (i.e. a heteroatom). Heterocycles are a reoccurring motif in organic chemistry and provide the core scaffold for many pharmaceuticals. One way to synthesize heterocycles is by using molecules containing an oxime, -C=N-OH, or oxime ether, -C=N-OCH3, functional group. Traditionally, such reactions are performed using metal catalysts or harsh reaction conditions. Recent experimental work in the de Lijser laboratory at CSUF has focused on developing a new, more environmentally friendly approach towards synthesizing heterocycles using oxime ethers containing a built-in nucleophile, light, and an organic catalyst. Specifically, photo-induced electron transfer is used to produce oxime ether radical cation reactive intermediates that subsequently undergo intramolecular cyclization. In collaboration with the de Lijser lab, we use computational chemistry, specifically density functional theory, to understand the reaction mechanism and explain trends in the experimental percent yields. For example, oxime ethers containing a thiophene group show very high yields of cyclized product whereas oxime ethers containing a pyridine group exhibit little to no reaction. Our computational results explain this trend by indicating that the pyridine containing compounds have a higher barrier for cyclization, decreasing the rate of the reaction, as well as a higher ionization potential, making oxidation of the oxime ether to form the radical cation less favorable. Our results also explain why the cyclization reactions are regioselective, with the newly formed C-N bond occurring adjacent to the heteroatom. Finally, we will present preliminary results of using calculated ionization potentials to screen a large family of oxime ethers for their propensity to undergo radical cation cyclization. By shifting focus from compounds under current experimental investigation to derivatives of these compounds containing additional electron-donating or electron-withdrawing groups, we will use computational chemistry to make predictions about reactions that have not yet been experimentally explored. In conjunction with the experimental work in the de Lijser group, this research will hopefully lead to an environmentally friendly and effective approach towards synthesizing the core scaffolds of a wide variety of pharmaceuticals.
Poster #: 204
Campus: CSU Los Angeles
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Immunostaining, mucosa, Innate immunity
Project Title: Visualization of Human Intestinal Epithelium and Immune Cell Markers Using Immunofluorescence & Immunohistochemistry
Author List:
Silva, Elvia; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Banuelos, Paula ; Undergraduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Gonzalez, Mildred Gonzalez; Undergraduate, Biological Sciences, California State University, Los Angeles
Paher, Bapi; Tulane University
Porter, Edith; Faculty, Biological Sciences, California State University, Los Angeles
Abstract: The human intestinal mucosa faces the challenge of absorbing nutrients while excluding pathogens. The small intestinal epithelium contains two regions, the villi and the crypts. The cells in both regions adhere to each other via tight junction proteins such as zonula occludens 1, or ZO-1. Various cell types constitute the intestinal epithelium, with the crypts housing the intestinal stem cells (ISC identified by their unique marker Lgr5). The Lgr5+ ISC differentiate via transit-amplifying cells into five cell lineages: enteroendocrine cells, goblet cells, enterocytes, the recently discovered tuft cells, and Paneth cells (PC). PC provide defense to the intestines in the form of secreted antimicrobial peptides such as human defensin 5 (HD5). However, PC have also been recently implicated in the regulation of the environmental niche for ISC by providing growth signals. We aim to investigate how infectious or inflammatory diseases of the small intestine affect the PC-Lgr5+ cross talk and epithelial cell integrity. Our objective was to establish protocols to simultaneously stain for ISC, PC, ZO-1, and activated caspase 3 to visualize the frequency and localization of these components. Antimicrobial peptides and tight junctions stain easily with fluoroprobe labeled antibodies with a three-color immunofluorescence protocol with DAPI nuclear counter stain. In contrast, activated caspase 3 and Lgr5+ signals require robust signal amplification via alkaline phosphatase and nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolyphosphate (BCIP/NBT) chromogen in an immunohistochemistry protocol with methyl green counter stain. Highlighting the localization and frequency of these markers will allow better understanding of PC-Lgr5+ ISC cross talk and epithelial dysbiosis in intestinal inflammation. Funding for this study was provided by RO1 grant #:1R01DK109883-01A1 and from the Louis Stokes Alliance for Minority Participation Bridges to the Doctorate Program at Cal State LA. The funding for this program is provided by the National Science Foundation under grant #HRD-1700556. Funding also provided by the National Institutes of Health, #NIH MBRS-RISE GM061331.
Poster #: 205
Campus: San Diego State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: in-vivo human calf muscle , medical imaging, magnetization transfer magnetic resonance imaging
Project Title: Magnetization transfer Magnetic Resonance imaging of Human Calf Muscle by Quantitative and Semi-quantitative methods: Gender and Regional Differences.
Author List:
Romero, Ignacio ; University of California at Merced
White, Cameron; Graduate, Physics, San Diego State University, Presenting Author
Sinha, Usha; Faculty, Physics, San Diego State University
Abstract: Quantitative magnetization transfer (qMT) enables the evaluation of the macromolecular content of tissue by selective saturation of bound protons at several offset frequencies and power of the MT pulse. Magnetization Transfer Saturation (MTsat) derives a semi-quantitative index of magnetization transfer in faster times than qMT. The macromolecular fraction and MTsat values may potentially reflect the collagen content of the muscle extracellular matrix. This study reports measurements of qMT and MTsat of skeletal muscle to investigate regional and gender based differences in calf muscle.
Eight young female/ male subjects were scanned on a 3T (Siemens, PRISMA) scanner. A 3D fast low angle shot (FLASH) sequence was used to acquire the MT-, T1- and proton density-weighted sequences to compute MTsat of the calf muscle. Data for the qMT was acquired with the 3D FLASH sequence varying the MT radiofrequency pulse offset and power (frequency range: 1- 50 kHz, flip angles: 3000, 5000).
Significant differences were seen between the male and female cohorts with MTsat being higher in males compared to the females. The higher MTsat values in the tibialis anterior (TA) muscle compared to the plantarflexor muscles was statistically significant. The analysis and calculation of the qMT maps are currently underway.
Regional differences in MTsat may potentially reflect fiber type; Type I fibers show a greater MT effect; it is likely that the higher collagen content in Type I fibers are the reason for the higher MT sat values in TA muscle. Gender based differences in MTsat values are harder to explain but may be related to the known smaller, higher density fibers in females leading to a smaller extracellular volume fraction.
Poster #: 206
Campus: CSU Los Angeles
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Parkinson’s disease , scanning ion conductance microscopy, surface plasmon resonance microscopy
Project Title: Real-Time Monitoring of α-Synuclein-Induced Cell Membrane Disruption Using Scanning Ion Conductance and Surface Plasmon Resonance Microscopies
Author List:
Parres-Gold, Jacob; Undergraduate, Chemistry and Biochemistry , California State University, Los Angeles, Presenting Author
Wong Su, Stephanie ; Undergraduate, Chemistry and Biochemistry , California State University, Los Angeles
Chieng, Andy; Undergraduate, Chemistry and Biochemistry , California State University, Los Angeles
Wang, Yixian ; Faculty, Chemistry and Biochemistry , California State University, Los Angeles
Abstract: Parkinson’s disease (PD) affects more than one million people in the United States alone, making it the second most common neurodegenerative disorder in the US. The misfolding and aggregation of alpha-synuclein (α-Syn), a 140-residue soluble protein populated at presynaptic termini, is believed to lead to high neurotoxicity and plays an important role in the development and progression of PD. One hypothesis is that α-Syn aggregates disrupt the cell membranes by forming pores that lead to permeabilization of lipid bilayers, eventually causing cell death. However, no work has been done to directly observe the pore formation at live cells. In this work, scanning ion conductance microscopy (SICM) was used to real-time monitor the morphological changes of live SH-SY5Y neuroblastoma cell membranes before and after the addition of α-Syn oligomers. Significant damage to the cell membrane was observed, with both large defects and small pore-like structures appearing after α-Syn aggregates were added to the media. Surface plasmon resonance microscopy (SPRM) was also used to monitor the binding of the α-Syn oligomers to the cell membrane. The use of SPRM in conjunction with SICM has allowed for the real-time observation of distinct binding events that correspond to observed damage. In addition, SPRM was used to calculate the value of α-Syn’s membrane binding affinity (KD) to compare the activities from α-Syn oligomers prepared in various conditions. Beforehand, those oligomer solutions were characterized by atomic force microscopy (AFM) and circular dichroism (CD) spectroscopy to confirm that oligomers were the dominant species present. Overall, this work has used SICM and SPRM to monitor α-Syn oligomer-induced disruption and perforation of neuroblastoma membranes in real-time, suggesting one means by which α-Syn oligomers may induce neuronal death in PD. This research was supported by the CSULA Startup Fund, CSUPERB New Investigator Grant, and the National Institute of General Medical Sciences of the National Institute of Health under Award Number R25GM061331.
Poster #: 207
Campus: CSU Fresno
Poster Category: Diagnostics/Imaging/Analytical
Keywords: x-ray fluorescence, breast cancer, spheroid culture
Project Title: Analyses of Trace Element Composition in a Metastatic Breast Cancer Model using X-Ray Fluorescence
Author List:
Pyreddy , Sagar; Undergraduate, Biology, California State University, Fresno, Presenting Author
Senthilvelan , Jayasuriya ; University of California, Los Angeles
Gherase, Mihai; Faculty, Physics, California State University, Fresno
Bush, Jason; Faculty, Biology, California State University, Fresno
Abstract: Trace elements have been reported as an accurate indicator of cancer cell proliferation in previous studies. To test whether differences in trace element composition would be an accurate indicator of metastatic behavior for breast cancer cells, we utilized X-ray Fluorescence (XRF). Variants of the common breast cancer cell line, MDA-MB-231, with differential metastatic potential to bone (MDA-MB-231P vs MDA-MB-231Bone) were used in a spheroid culture system as a surrogate environment for in vivo growth. A novel spheroid harvesting method was designed and developed for sample placement on a kapton tape holder. XRF spectra were analyzed with MATLAB and a combination of operations: (i) a low pass Butterworth filter, (ii) Gaussian models were then fit to each elemental peak, (iii) Bremsstrahlung intensity corrections were performed on the amplitudes, and (iv) two sample Student t-tests were performed on the estimated Gaussian amplitudes to calculate the statistical significance in elemental composition difference between MDA-MB-231P and MDA-MB-231Bone. All trials found that the osteotropic cells consistently accumulated more Fe and Pb (p-value of < 0.0001). As opposed to the current gold standard of histopathology, XRF is quantitatively objective, relatively inexpensive, and extremely fast. The detected differences are proof-of-concept that XRF could be reliably used as a clinically-significant indicator of metastatic potential or tumor stage.
Poster #: 208
Campus: CSU San Bernardino
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Assay development, Peroxisome, Luciferase
Project Title: Development of a novel biochemical assay for peroxisome biogenesis and cargo transport
Author List:
Vuong, Chau; Undergraduate, Chemistry, California State University, San Bernardino, Presenting Author
Nickerson, Daniel; Faculty, Biology, California State University, San Bernardino
Abstract: Peroxisomes are membrane-bound organelles that support numerous biochemical functions, including breakdown of long-chain fatty acids and synthesis of lipids critical to nervous and cardiovascular functions. Defects in peroxisome biogenesis or maintenance are linked to a broad array of human diseases such as lipid metabolism disorders and neurodegenerative diseases. Peroxisomes proliferate either through fission of existing peroxisomes, or cells can build new peroxisomes de novo from vesicles that bud from the endoplasmic reticulum (ER) to deliver lipid and protein cargos to peroxisomes. We report our efforts to develop a novel, luciferase-based biochemical assay of peroxisome biogenesis and cargo transport.
Our assay involves an enzyme reporter, Gaussia luciferase (GLuc), split into two halves (GLucN & GLucC) that lack light-emitting activity on their own, but that assemble together into a functional enzyme. We fused GLucC to the luminal terminus of a peroxisome transmembrane protein (Pex3). When GLucC-Pex3 is transported from the ER to a peroxisome, the GLucC tag faces the lumen of the peroxisome. The other half of the reporter, GLucN, was tagged at either the amino- or carboxy-termini with peroxisome import signals that allow GLucN to enter the peroxisome lumen and assemble with GLucC-Pex3.
Assay optimization and control experiments in peroxisome-replete cells revealed that all engineered and sequence-verified GLuc reporter constructs expressed in yeast and were detectable at expected molecular weight via Western blot. As expected, splitting the GLuc enzyme into cytosolic halves resulted in a ~98% reduction in luciferase signal compared to cytosolic, full length GLuc. Unexpectedly, targeting both soluble GLucN and GLucC halves to the peroxisome lumen resulted in a further 10-fold reduction in signal, indicating that the peroxisome lumen might be a non-permissive environment for GLuc. Fusing GLucC to the luminal terminus of Pex3 reduced signal ~10-fold further, a level barely above background. Phenotypic assays indicated that the chimeric GLucC-Pex3 reporter failed to rescue the oleic acid growth defect of cells lacking Pex3, indicating that the GLucC tag disrupts function of Pex3. Future directions include constructing a new chimeric reporter using the transmembrane protein Pex22, as well as examining alternative reporter enzymes. This work was supported by a Summer Undergraduate Research Program grant from CSUSB Office of Student Research.
Poster #: 209
Campus: CSU Los Angeles
Poster Category: Diagnostics/Imaging/Analytical
Keywords: 3D-microfluidics, glucose assay, colorimetric assay
Project Title: 3D Multilayered Paper- and Thread/Paper-Based Microfluidic Devices for Bioassays
Author List:
Neris, Natalia M.; Graduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Guevara, Ricardo; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles
Gonzalez, Ariana; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles
Gomez, Frank A.; Faculty, Chemistry and Biochemistry, California State University, Los Angeles
Abstract: A current global challenge is the increasing cost of healthcare, putting populations at risk due to hospital care limitations. Microfluidic technologies have been successfully incorporated in point-of-care (POC) testing thereby reducing hospital visitations and costs by bringing treatment directly to primary care clinics. POC diagnostic devices incorporating microfluidic platforms require microliter volumes of fluid from a patient thereby increasing the gamut of tests available to a clinician. Paper-based devices have been shown to be a viable alternative to traditional laboratory techniques due to their ease of fabrication, low cost, and low sample volume requirements. Herein, we describe the fabrication of novel three-dimensional multilayered microfluidic paper-based analytical devices (3D-µPADs) and a 3D microfluidic thread/paper-based analytical device (3D-μTPAD) to detect glucose and bovine serum albumin (BSA) through colorimetric assays. The 3D-μPAD and 3D-µTPAD consisted of three (wax, heat pressed wax-printed paper, single-sided tape) and four (hole-punched single-sided tape, blank chromatography circles, heat-pressed wax-printed paper, hole-punched single-sided tape containing trifurcated thread) layers, respectively. The saturation curves for each assay were generated for all platforms. For the glucose assay, a solution of glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI) was flowed through each platform and, upon contact with glucose, generated a yellow-brown color indicative of the oxidation of iodide to iodine. For the protein assay, BSA was flowed through each device and, upon contact with citrate buffer and tetrabromophenol blue (TBPB), resulted in a color change from yellow to blue. The devices were dried, scanned, and analyzed yielding a correlation between either yellow intensity and glucose concentration or cyan intensity and BSA concentration. A similar glucose assay, using unknown concentrations of glucose in artificial urine, was conducted and, when compared to the saturation curve, showed good correlation between the theoretical and actual concentrations (percent differences < 10%). The use of 3D-based devices incorporating paper and thread for POC diagnostic devices is a viable alternative to more expensive microfluidic-based patterning techniques. The ease of fabricating 3D MDs demonstrates the potential for developing other devices and for multiple analytes simultaneously.
Poster #: 210
Campus: Cal Poly Pomona
Poster Category: Diagnostics/Imaging/Analytical
Keywords: aptamers, qPCR, diagnostics
Project Title: Optimization of the Selection and Screening of DNA Aptamers
Author List:
Garrett, Michael; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Chen, Andrew; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Liu, Junjun; Faculty, Biological Sciences, California State Polytechnic University, Pomona
Abstract: DNA aptamers are short, single-stranded oligomers capable of binding to a wide variety of target molecules with high affinity and specificity. DNA aptamers have emerged as ideal alternatives to antibodies in diagnostics due to their low cost of production, long shelf-life, and high stability. The selection of specific aptamers involves a process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment). During this process, a library of DNA aptamers consisting of approximately 1014 unique random sequences are enriched until the pool is made up of mainly the unique aptamers which possess affinity to the target molecule. While aptamers possess significant advantages over current antibody-based testing, they currently require a relatively large upfront labor and monetary investment. First, by modifying and optimizing several steps of SELEX, we have developed a robust and cost-effective method of aptamer selection. For the generation of single stranded DNA aptamers, we tested three of the most common methods in current literature and found lambda exonuclease digestion to be the most effective and robust. Second, during the optimization of the washing steps, a series of wash buffers with increasing salt concentrations were tested. We found that a PBS-T wash buffer containing up to 1.5M NaCl provided a high level of ionic stringency without causing the target protein to dissociate from the membrane it was immobilized on. Third, in addition to improvements made to the enrichment process of SELEX, we employed a qualitative aptamer screening method by using qPCR (Quantitative Polymerase Chain Reaction). Enriched aptamers were cloned into vectors that allowed us to isolate clone colonies containing a single aptamer sequence. The qPCR allowed us to quickly validate the affinity of individual aptamers to target molecules in a cost effective way. Using this method, we were able to identify two promising aptamers with particularly high affinity for our target molecule, which was a Listeria monocytogenes surface protein. In conclusion, the protocol developed presents a cost effective and robust method of selecting DNA aptamers to be used in diagnostics. This project was supported by a grant from California State University Agriculture Research Institute (ARI).
Poster #: 211
Campus: Cal Poly San Luis Obispo
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Nanoscale manipulation, Enzyme stability, Lyophilization and Storage
Project Title: Reducing Waste of Biological Reagents: Polymer-Based Solid Dispersions of Enzymes
Author List:
Mitchell, Megan; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author, Nagel Award Nominee
Strong, Brandon; Graduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Plastina, Gabi; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Escamilla, Emiliano; UCSD
Martinez, Andres; Faculty, Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo
Martinez, Nathaniel; Faculty, Biological Sciences, California Polytechnic State University, San Luis Obispo
Abstract: Background: The primary objective of this project is to develop a cost-effective, reliable method for manipulating and preserving valuable laboratory reagents, thus leading to increased savings and decreased biological waste for scientific laboratories, pharmaceutical companies, and other industries. A key 2015 study in PLoS Biology stated that 36% of wasted research expenses are assigned to biological reagents, which results in losses of greater than $10 billion annually in the United States alone. Therefore, innovations to reduce costs associated with wasted reagents are critically necessary.
An inherent limitation of protein manipulation is the chemical instability of a protein’s molecular structure, especially when stored in solution. To combat this limitation, proteins are often lyophilized (a.k.a, freeze-dried). Proteins in solution are highly manipulable (i.e., dilution via pipette) yet inherently unstable, and lyophilized proteins are highly stable but cannot be manipulated easily. Our proposed method of polymer-based solid dispersion may allow for both simultaneously.
Methods: Horseradish peroxidase (HRP), alkaline phosphatase (AP), and quinine hydrochloride were examined as model reagents for dispersion. Reagents and polyethylene glycol (PEG) were added in solution to a tube at low ratios (1:10,000-1:1,000,000) prior to being lyophilized (-50C). PEG was chosen as the polymer dispersant due to its inertness, high solubility, low cost, and use in many pharmaceuticals. Small amounts of the resulting homogenous mixtures were rehydrated and tested for dispersion variability and manipulation thresholds.
Results: There was no significant difference between reagent alone (diluted via pipette), dispersions rehydrated, or the reagent with equivalent PEG added after for 50 ng/mL HRP (f=0.857, df=2,5, p=0.479), 50 ng/mL AP (f=1.803, df=2,5, p=0.257), or 10 ng/mL quinine (f=0.223, df=2,9, p=0.804). Dispersions were rehydrated in 1 mL, indicating manipulation of 50 ng of enzyme and 10 ng of quinine in solid state, using standard laboratory balances.
Conclusion: We successfully manipulated as low as 50 ng of enzyme in solid form, thereby maintaining stabilization and preserving valuable reagents. This novel method of protein manipulation has the potential to significantly reduce waste (and associated cost) of precious reagents by allowing for nanoscale manipulation and increased ambient reagent stability.
Poster #: 212
Campus: San Diego State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: chromaTOF, PyAutoGUI, contaminants
Project Title: Automation of chromaTOF and software development for non targeted analysis of contaminants
Author List:
Sharma, Richa; Graduate, Bioinformatics, San Diego State University, Presenting Author
Kelley, Scott; Faculty, Biology, San Diego State University
Hoh, Eunha; Faculty, School of Public Health, San Diego State University
Dodder, Nathan; Faculty, School of Public Health, San Diego State University
Richardot, William; Staff, San Diego State University Research Foundation, San Diego State University
Abstract: Non-targeted analysis of water pollutants is of paramount importance as it can identify novel contaminants that could potentially cause deleterious biological effects. The available mass spectral libraries may miss a significant number of these novel contaminants. Hence, we cannot depend chiefly on these libraries for identification of these compounds. However, the non-targeted analysis yields huge volumes of data, which can be extremely difficult to analyze manually. Hence, the goal of this project is to develop a custom program to evaluate this data, using python as the primary coding language.
In the first phase of the project, we targeted at automating the extraction of mass spectra information in NIST format from the chromaTOF software used for analyzing the mass spectral data. This was achieved using PyAutoGUI, which is a python module to control the use of mouse and keyboard for automation of any Graphical User Interface. PyAutoGUI can reproduce human actions like moving, clicking and dragging the mouse, pressing and holding keys, and also pressing keyboard hotkey combinations. As a result of this, we can now successfully extract the mass spectra information for analysis in a significantly reduced time with negligible human effort.
In the second phase of the project, we aim to develop a robust program for calculating a similarity score between unknown compounds from our samples and known compounds from NIST library. A scoring scheme is designed to calculate the similarity scores, which is based on the presence of three most prominent ions, the presence of the molecular ion in the peak mass spectra and percentage of ions that present in both sample and NIST library hit mass spectra. We are currently coding for this program and the results of the first and the second phase of the project will be presented at the symposium.
Poster #: 213
Campus: Stanislaus State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Spectroscopy, Corrole, Electrochemistry
Project Title: Photophysical, Electrochemical, and Spectroelectrochemical Characterization and Solvent Effect on the Tautomerism of Free-Base Corrole
Author List:
Webb, Kevin; Undergraduate, Biology, California State University, Stanislaus, Presenting Author
Hernández, Vanessa; Undergraduate, Computer Science, California State University, Stanislaus, Presenting Author
Reed, Christopher; Undergraduate, Chemistry, California State University, Stanislaus
Alemán, Elvin; Faculty, 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.
We investigated the photophysical, electrochemical, and spectroelectrochemical properties of free-base triphenyl corrole (H3TPCor). We have characterized corroles ability to absorb light in a series of 10 solvents and in a multitude of concentrations (aggregation effect). We have also characterized the excited state emission of H3TPCor and the role of solvent in the lifetime of the same excited state. UV-Visible data shows a trend indicating that the transition from one tautomer to another is dominated by the H-bonding donating ability of the solvent molecules with the core of H3TPCor. We have also found that both tautomers can co-exist in some of the solvents, and we have built a model to estimate the amount of each tautomer and what solvent property better correlate with this trend. One tautomer shows four oxidation potentials in the electrochemistry experiments, while the other shows three oxidation potentials. Both tautomers have only one reduction potential. UV-Visible spectra of reduced H3TPCor species all followed the same trend. The results indicate that the reduced species prefers this form irrespective of the solvent used. When oxidized, H3TPCor spectrum resembles the profile of the most stable tautomer.
Poster #: 214
Campus: CSU Fresno
Poster Category: Disease (Pathogens)
Keywords: Bacillus, probiotics, antibiotic resistance
Project Title: Antimicrobial Resistance in Bacillus Strains Found in Probiotics
Author List:
Flores, Emmanual; Graduate, Biology, California State University, Fresno, Presenting Author
Kaze, Mo; UC Merced
Sistrom, Mark; UC Merced
Van Laar, Tricia; Faculty, Biology, California State University, Fresno
Abstract: Probiotics are marketed to humans and domesticated animals globally as providing therapeutic/protective effects against gut microflora imbalances. Probiotics are typically consumed in food or as supplements. Despite the pathogenic characteristics of some Bacillus spp. (B. anthracis, B. cereus), B. subtilis and B. coagulans are deemed “generally recognized as safe” (GRAS) by the FDA and are added to a plethora of probiotic products. Although these species have attained GRAS status, they may still possess genes conferring antimicrobial resistance (AMR) which could be shared among other bacteria. In this study, we aim to determine if probiotic Bacillus are resistant to antibiotics and analyze their potential to transfer AMR genes to potentially pathogenic Bacillus. We tentatively identify commercial Bacillus strains through gram and endospore staining before confirming with 16S rRNA gene sequencing. Once confirmed as Bacillus, we perform antibiotic sensitivity assays on each strain using a disk-diffusion method with a panel of 15 antibiotics. To date, we have isolated and tested six strains isolated from chicken feed, kombucha, kimchi, and root beer and found resistance to antibiotics such as bacitracin and sulphamethizole. We will perform whole genome sequencing of all strains not already deposited in NCBI. The second part of this study is using bioinformatic analyses to mine sequenced genomes of B. subtilis and B. coagulans for the presence of known antibiotic resistance cassettes. We downloaded all draft and assembled genomes of B. subtilis (170) and B. coagulans (28) and compared them with the Comprehensive Antibiotic Resistance Database (CARD) using BLAST command line tools. We found resistance to macrolides, lincosamides, streptogramines (MLS) in the B. coagulans sequences. In B. subtilis, we found even more AMR genes conferring resistance to MLS, beta-lactams (including carbapenems), fluoroquinolone, rifampin, tetracycline, and aminoglycosides, among others. Finally, as Bacillus species are able to readily engage in horizontal gene transfer with other members of the genus, we will perform mating assays to determine whether some of these AMR genes can be transferred to more pathogenic species, including B. cereus or B. pumilus. Taken together, the results from this study will provide insight into the potential risks of antimicrobial resistance in probiotic supplements and the potential for spread of AMR to pathogenic species.
Poster #: 215
Campus: CSU Los Angeles
Poster Category: Disease (Pathogens)
Keywords: Candida albicans, Antifungal agent, pyrvinium pamoate
Project Title: Pyrvinium pamoate inhibits Candida albicans growth and morphogenesis
Author List:
Zuluaga, Cipriano; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Macias, Marlene; Graduate, Biological Sciences, California State University, Los Angeles
Park, Hyunsook; Faculty, Biological Sciences, California State University, Los Angeles
Abstract: Candida species are the fourth most common cause of bloodstream and cardiovascular infections in the US. Moreover, Candida related blood infections are more frequent in neonatal care units and cause serious morbidity and mortality. Individuals with bloodstream infections caused by Candida sp. have a 50% mortality rate even with treatment. However, treatment is limited due to a lack of effective antifungal agents available for clinical usage. Thus, it is critical to find new antifungal drugs that are effective against Candida albicans. We tested Pyrvinium pamoate (Pyr), an FDA approved anthelmintic drug, for its effect on the growth and morphogenesis of C. albicans, the latter being important for biofilm formation and host cell adherence. Our results suggest that Pyr inhibits the planktonic growth of C. albicans in a dose dependent manner. After the 12-hour time point the cells treated with 0.25μg/ml of Pyr had a 21% reduction, cells treated with 0.5 μg/ml of Pyr had a 31% reduction, cells treated with 1.0 μg/ml of Pyr had a 56% reduction, cells treated with 2.5 μg/ml of Pyr had a 76% reduction, and cells treated with 5.0 μg/ml of Pyr had an 87% reduction. Moreover, the yeast cells’ budding pattern was abnormal and size was diminished as the concentration of Pyr increased. Similarly, germ tube formation was severely affected at 5.0 μg/ml, indicating that Pyr inhibits morphogenesis of C. albicans. The calcofluor white (CFW) staining in C. albicans showed that chitin accumulated on the cell wall of both yeast and hyphal cells treated with 2.5 µg/mL and 5.0 µg/mL of Pyr, suggesting that Pyr perturbs cell wall integrity. This study demonstrates that Pyr has a significant inhibitory effect on C. albicans growth and morphogenesis. Future studies will test Pyr’s mode of action and cellular targets to facilitate the development of pyrvinium pamoate-based antifungal drugs.
Poster #: 216
Campus: CSU East Bay
Poster Category: Disease (Pathogens)
Keywords: Toxoplasma, virulence, subcellular localization
Project Title: How does ROP23 contribute to Toxoplasma pathogenesis?
Author List:
Rodriguez, Paula; Undergraduate, Biological Sciences, California State University, East Bay, Presenting Author
Guiton, Pascale; Faculty, Biological Sciences, California State University, East Bay
Abstract: Toxoplasma gondii is an intracellular Apicomplexan responsible for the zoonotic disease toxoplasmosis. T. gondii utilizes the contents of specialized secretory organelles at its apical end to exert its virulence. Specifically, T. gondii injects proteins (ROPs and RONs) from the rhoptry organelles directly into its host during invasion. While many rhoptry proteins have been implicated in the parasite’s pathogenic processes, only a handful have been fully characterized to date. My project focuses on the putative rhoptry protein, ROP23. Though present in all three infectious forms of the parasite, tachyzoites, bradyzoites and sporozoites, the expression of rop23 is significantly increased during the chronic stage of toxoplasmosis. Understanding the contributions of this protein will provide further insight into the mechanisms T. gondii and other Apicomplexans employ for cellular invasion and for immune modulation once in the infected cell.
We have created a T. gondii parasite line defective in rop23 using the CRISPR/Cas9 gene editing system. This ablation renders T. gondii avirulent in a murine model of toxoplasmosis. Given the presence of a signal peptide and its potential role in virulence, I postulate that ROP23 localizes to the rhoptry organelles of T. gondii and it is directly secreted into the host where it modulates host processes. We will generate a mutant strain of T. gondii that expresses an epitope-tagged version of ROP23 and perform immunofluorescence assays for localization studies during infection. Thus far, we have generated the plasmid that will allow the integration of a hemagglutinin A (HA) tag sequence at the C-terminus of ROP23. We have begun transfection into T. gondii. Upon completion of this project, we will gain a better insight into the localization of ROP23 in T. gondii during its invasion, which will generate new hypotheses as to the function of this protein during the pathogenic process. Findings from this work, which may be applicable to other members of the phylum Apicomplexa, which includes Plasmodium spp. and Cryptosporidium spp., can provide novel targets for prevention and treatment of diseases associated with these parasites.
Poster #: 217
Campus: CSU San Bernardino
Poster Category: Disease (Pathogens)
Keywords: Influenza virus , epitope tag, protein interactions
Project Title: Generation of Influenza NP-FLAG virus to facilitate NP interaction studies
Author List:
Berumen , Joscelyn ; Undergraduate, Biology , California State University, San Bernardino, Presenting Author
Rodriguez , Raquel ; Undergraduate, Biology , California State University, San Bernardino, Presenting Author
Newcomb, Laura ; Faculty, Biology , California State University, San Bernardino
Abstract: Influenza A viruses cause seasonal epidemics of respiratory illness and are responsible for a large health and economic burden annually. Influenza A is combated with annual vaccinations that have variable efficacy due to the rapidly changing nature of the virus, and antiviral therapies that overtime become ineffective as resistance is selected. This underscores the need to identify novel targets for new antiviral therapies. The influenza viral ribonucleoprotein (vRNP) is responsible for influenza RNA synthesis and highly conserved across multiple influenza subtypes, making this an excellent antiviral target. Nucleoprotein (NP) is an essential component of vRNPs and interacts with both viral and host factors to regulate viral RNA expression. We aim to examine NP protein interactions during the viral life cycle through generation of influenza A virus expressing an NP-FLAG tagged protein. The 7-amino acid FLAG epitope tag will allow us to use commercially available antibody against the FLAG tag to isolate and visualize the NP protein. Previous results in our lab concluded addition of the FLAG coding sequence disrupted the NP vRNA packaging signal, which overlaps with NP coding sequences. Attempts to generate this virus were unsuccessful. Here we constructed a DNA plasmid to express NP-FLAG coding sequence with an additional repeat of RNA containing packaging signals (PS) reported to result in 60% packaging efficiency of the NP vRNA segment. Human tissue culture cells (293T) were transfected with 12 DNA plasmids required to generate Influenza A virus and 24 hours later overlaid on MDCK cells to amplify virus. Samples were collected at different time points post overlay and tested for virion presence in hemagglutination assay (HA). While we obtained WT-NP virions, we did not detect virions from NP-FLAG-PS. To confirm the NP-FLAG-PS vRNA is recognized as a template in the vRNP complex and produces a functional NP-FLAG protein, a vRNP reconstituted assay was performed. 293T cells were transfected with or without the viral polymerase and NP-FLAG-PS vRNA template. Western blot results reveal robust NP-FLAG expression in the presence of the viral polymerase, indicating the NP-FLAG-PS vRNA forms a vRNP which results in functional NP-FLAG protein. We will attempt to improve WT-NP virus yield to recover NP-FLAG-PS virus. If successful, generate of NP-FLAG expressing influenza A virus will provide a tool for the influenza community to study NP and NP interactions.
Poster #: 218
Campus: Cal Poly San Luis Obispo
Poster Category: Disease (Pathogens)
Keywords: Escherichia coli, glycogen, urinary tract infection
Project Title: Escherichia coli strains that accumulate glycogen display increased growth in urine
Author List:
Andrews, Kaylee; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Yep, Alejandra; Faculty, Biological Sciences, California Polytechnic State University, San Luis Obispo
Abstract: Uncomplicated urinary tract infections (UTIs) are the second most common infection after respiratory tract ones, affecting more than 50% of otherwise healthy women. UTIs begin when bacteria normally residing in the digestive tract ascend through the urethra and colonize the bladder. In 75-90% of cases, UTIs are caused by uropathogenic strains of Escherichia coli. Glycogen is a glucose polymer that E. coli cells can accumulate when entering stationary phase or under nutrient depletion as long as a carbon supply abounds. While the exact role of glycogen storage in bacteria is not fully understood, it may serve as a source of carbon and energy in poor nutritional conditions. We hypothesized that E. coli strains that are capable of accumulating glycogen may have a growth advantage when transferred to carbon-poor media, specifically urine. To test this, we grew a panel of ~150 diverse E. coli strains from different origins in Kornberg medium (KM) agar. KM is a rich medium supplemented with glucose that promotes bacterial glycogen accumulation. After overnight growth, cells were stained with iodine, which reacts with glycogen giving a brown color. We categorized strains according to their ability to accumulate glycogen, ranging from 0=white=no glycogen accumulation to 3=dark brown=highest glycogen accumulation. About 10% of all strains scored a 3 and were selected for further experiments. Strains were grown overnight in KM or Luria-Bertani broth, which is also a rich medium but does not promote glycogen accumulation. Cells were washed in PBS and transferred to sterile human pooled urine. Growth curves in urine were constructed by recording absorbance at 600nm for 5h. Urine is a low nutrient medium and cells typically display poor growth and a significant lag phase when transferred from rich media. We show that, for most isolates, the lag phase in urine was shorter and the final cell density higher when strains had been grown overnight in KM compared to the same strains grown overnight in LB. The shortening of the lag phase and the more robust growth suggest that glycogen accumulation could play a role in uropathogenesis by supporting initial colonization of the bladder by E. coli. Additionally, the results of our glycogen accumulation survey show that there is great variation in the ability of E. coli strains to accumulate this polysaccharide.
Poster #: 219
Campus: CSU Sacramento
Poster Category: Disease (Pathogens)
Keywords: Polymicrobial, Metabolites, Wounds
Project Title: Cooperation and Competition for Short and Long Chain Fatty Acid Metabolites within Polymicrobial Biofilm Infections
Author List:
Hua, Thy; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Nguyen, Gloria; Graduate, Biological Sciences, California State University, Sacramento, Presenting Author
Cummings, Ryan; Graduate, Biological Sciences, California State University, Sacramento
Davis, Tyler; Graduate, Biological Sciences, California State University, Sacramento
Crawford, Robert; Faculty, Biological Sciences, California State University, Sacramento
Abstract: Persistent polymicrobial infections impede the treatment and healing of chronic wounds by forming multi-species communities called biofilms. Biofilm formation is highly regulated and culminates in the production of a self-initiated extracellular matrix (ECM), and these population-dense communities prolong tissue inflammation and provide resistance to antibiotics. Thus, a critical barrier to wound healing is the paucity of non-antibiotic therapeutics that inhibit microbial growth and prevent biofilm development. Short and long chain fatty acid metabolites modulate metabolism of yeast and bacteria in mixed populations and alter inflammatory mediators within infected tissue sites, yet the underlying mechanisms of microbial cooperation and competition for limited nutrient sources within bacterial communities remains largely uncharacterized. Here we show that long chain fatty acid metabolites purified from supernatants of pathogenic Malassezia restricta (MR, yeast) growth media, in turn, enhanced metabolic rates and biofilm formation of pathogenic Staphylococcus aureus (SA, bacteria) but were inhibitory for those phenotypes to commensal Staphylococcus epidermidis (SE, bacteria). On the other hand, short chain fatty acid metabolites purified from SE growth media, in turn, reduced metabolic rates and biofilm formation of pathogenic MR and SA while mediating significant increases for those phenotypes to a new generation of clonally-derived commensal SE cells. Additionally, we developed DNA probes to visualize yeast and bacteria spatial dynamics in polymicrobial biofilms by confocal scanning laser microscopy and quantified these interactions using real-time PCR. Our results show that the relative abundance of each species could be shifted when exogenous free fatty acids of varying chain lengths were added or withheld in a concentration-dependent manner from the biofilm-forming media. Collectively, these results begin to differentiate roles for bacteria and yeast species and their associated metabolites in mediating polymicrobial biofilm communities that potentiate skin infections and prevent therapeutic intervention. These studies were supported by campus grants awarded by various departments at CSUS.
Poster #: 220
Campus: CSU Los Angeles
Poster Category: Disease (Pathogens)
Keywords: Acinetobacter baumannii LAC-4, siderophore, virulence factor
Project Title: The generation of a markerless twelve-gene cluster knockout for the hydroxamate siderophore cluster in hypervirulent Acinetobacter baumannii LAC-4.
Author List:
Leal, Alejandra; Undergraduate, Department of Biological Sciences, California State University, Los Angeles, Presenting Author
Ewing, Peter; Faculty, Department of Biological Sciences, California State University, Los Angeles
Xu, Howard; Faculty, Department of Biological Sciences, California State University, Los Angeles
Abstract: Acinetobacter baumannii is a Gram-negative opportunistic pathogen that is increasingly resistant to multiple classes of antibiotics, posing serious concerns in healthcare worldwide, particularly to immunocompromised patients. A. baumannii is mostly known for causing respiratory infections such as ventilator associated pneumonia, but it is also known to cause bloodstream infections, meningitis, and surgical site wound infections. Due to its prevalence and increasing multi-drug resistance, novel strategies to combat this pathogen are urgently needed. One such approach is the design and discovery of antivirulence therapeutics wherein virulence factors are targeted in order to reduce the pathogens’ infectious capacity. One key virulence factor that can be potentially exploited is A. baumannii’s ability to acquire iron from its hosts. In this study, we focus on a twelve-gene hydroxamate siderophore cluster (HDX) to assess the roles of the genes in this cluster on hypervirulence of A. baumannii LAC-4, a hypervirulent strain acquired from an outbreak in a Los Angeles County hospital. Our plan was to delete the entire HDX gene cluster using a markerless gene knock-out (KO) method established in our laboratory. First, a KO construct containing flanking regions of the gene cluster linked to an apramycin resistance marker was generated via overlap extension PCR. The construct was then ligated into a suicide vector pEX130 and the recombinant plasmid was transformed into LAC-4 by electroporation. Since the plasmid cannot replicate independently in LAC-4 cells, growing transformants on medium containing apramycin selects single crossover mutants in which the plasmid integrates into the chromosome at one of the regions flanking the HDX cluster through homologous recombination. Confirmed mutants (co-integrants) were passaged in sucrose containing medium under SacB based counter-selection, thereby enriching double-crossover mutants. Double-crossover mutants were screened via molecular tests. Ultimately, the markerless HDX gene cluster KO mutant was identified and confirmed by genomic analysis via PCR and Sanger sequencing. The mutant will be further investigated in vitro and in vivo to determine the role of HDX cluster in the hypervirulence of LAC-4.
Poster #: 221
Campus: CSU Los Angeles
Poster Category: Disease (Pathogens)
Keywords: Tuberculosis, Mucosal Immunity, Epithelial cells
Project Title: Augmenting the Barrier Function of Alveolar Type II Cells with the Cytokines Tumor Necrosis Factor-α, Interferon-γ, Interleukin-17, and Interleukin-22
Author List:
Coello, Daniel; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Porter, Edith; Faculty, Biological Sciences, California State University, Los Angeles
Abstract: Mycobacterium tuberculosis (Mtb) is the primary infectious agent that causes tuberculosis (TB). Mtb has infected approximately 1/4 of the world’s population and 10 % of the infected will develop active TB. Yet, the ability to treat the infection diminishes because of the emergence of multidrug resistant strains. The preventive measures against TB through vaccination involving the adaptive immune system are limited. Our laboratory is interested in investigating whether the innate mucosal host response could be exploited in novel therapeutic and preventative designs to combat TB. Epithelial cells lining the airways provide the first line of defense against airborne pathogens by forming a physical and chemical barrier. Airway epithelia release antimicrobial peptides (AMPs) such as human beta defensin-2 (HBD2), antimicrobial lipids (AMLs), and chemokines like IL-8 to attract other immune cells including T helper cells that are integral to the adaptive immune response. T-helper derived cytokines, part of the adaptive immune system, have been shown to be key player in the immune system’s ability to properly respond to a bacterial infection; therefore, it is possible that these cytokines can have an effect of airway epithelia. In this pilot study, A549 cells, alveolar type II epithelial cells, were grown in air-liquid interface and stimulated with cytokines secreted by T helper cells (5 ng/mL each): Tumor Necrosis Factor-α (TNF-α) & Interferon-γ (IFN-γ) and Interleukin-17 (IL-17) & Interleukin-22 (IL-22) for 4 h to induce AMPs and AMLs production. IL-8 and HBD2 gene expression was quantified by RT-PCR. Lipid secretion was assessed with Nile Red. The physical barrier integrity was assessed using fluoroprobe labeled phalloidin. The gene expression of IL-8 was upregulated 9-fold compared to the control when cells were stimulated with TNF-α & IFN-γ indicating that in our culture system A 549 cells were responsive to cytokines. Nile Red fluorescence of cell secretion was 3 times greater compared to the control when cells were stimulated with IL-17 & IL22 suggesting that T-helper derived cytokines can promote AML secretion by airway epithelia. Phalloidin staining was consistent with tight junction formation under all test conditions. This data suggests that T helper cell derived cytokines can directly augment the potential of airway epithelia to combat an airborne pathogen and that epithelial cells may be included in future vaccine design. NIH MBRS-RISE GM061331.
Poster #: 222
Campus: CSU Fullerton
Poster Category: Disease (Pathogens)
Keywords: Drosophila melanogaster, immunity, Beauveria bassiana
Project Title: Determining the tradeoffs between mating and immunity in male and female Drosophila melanogaster
Author List:
Ekanath Rai, Kshama; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Cheek, Madison; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Abstract: Fruit flies, Drosophila melanogaster, are powerful model organisms for studying immune defense as they share innate immunity with humans. Through molecular genetic experiments with D. melanogaster mutants, we previously showed that females are more susceptible than males to fungal infection and that this dimorphism is mediated by Toll and Imd pathway genes. Sexual dimorphism in infection resistance may be due to differential investment in reproduction after mating. Post-mating immunosuppression has been found to be predominantly a female trait in Drosophila when infected with four different bacterial pathogens. However, post-mating immunosuppression has not been studied with fungal pathogens. Here we tested the effects of mating on immune defense when introduced to the fungal pathogen Beauveria bassiana. Mortality assays and statistical analyses were conducted to determine differences in susceptibility between virgin and mated flies. Mated female flies and mated male flies had decreased survival rates when compared to virgin female flies and virgin male flies. Male and female flies that mated daily throughout adulthood showed lower survival than flies that mated only for one day. Our results suggest that post-mating immune suppression happens in both males and females, unlike previously thought. A robust post mating immunosuppression in Drosophila survival to B. bassiana presents opportunities to further dissect the underlying mechanisms with applications for biological control of insect vectors of human disease, insect crop pests, and also to elucidate pathways involved in human innate immune defense.
Poster #: 223
Campus: Cal Poly San Luis Obispo
Poster Category: Disease (Pathogens)
Keywords: non-alcoholic fatty liver disease , probiotics , PCR
Project Title: PROBIOTIC INCLUSION IN A HIGH-FRUCTOSE HIGH-FAT DIET DOES NOT PREVENT THE ONSET OF LIVER INJURY IN A PIG MODEL OF PEDIATRIC NON-ALCOHOLIC FATTY LIVER
Author List:
Hernandez, Gabriella ; Graduate, Animal Science, California Polytechnic State University, San Luis Obispo, Presenting Author
Melnyk , Megan; Undergraduate, Biological Sciences , California Polytechnic State University, San Luis Obispo, Presenting Author
Smith, Victoria; Graduate, Animal Science, California Polytechnic State University, San Luis Obispo
Columbus, Daniel; Prairie Swine Centre, Inc., Saskatoon, SK
VanderKelen, Jennifer; Faculty, Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo
Kitts, Christopher; Faculty, Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo
Burrin, Douglas; Baylor College of Medicine
Manjarin, Rodrigo; Faculty, Animal Science, California Polytechnic State University, San Luis Obispo
Maj, Magdalena ; Faculty, Biological Sciences , California Polytechnic State University, San Luis Obispo
Abstract: Probiotics have been shown to ameliorate the clinical signs of non-alcoholic fatty liver disease (NAFLD), a chronic liver disorder associated with excessive fat and carbohydrate consumption in children. Despite improvements in nutritional management of pediatric NAFLD, more than 4.5 million of children are expected to develop the pathology in the US next year. Improving our understanding of the mechanisms by which diet and probiotics regulate the gut-liver axis early in life has the potential to translate into practices that may improve pediatric health. In this study, 28 10-d old piglets were assigned to receive 1 of 4 liquid diets during 10 weeks: 1) Control (CON; n=8), 2) high-fructose high-fat (HFF; n=6), 3) CON supplemented with probiotic (CONP; n=6), and 4) HFF supplemented with probiotic (HFFP; n=6). Animals were fed 40 mL · kg BW-1 at 6-h intervals 4 times per day. Probiotics were added to the liquid diets at 6.2 × 104 cfu · mL-1 every 24 h to prevent bacteria overgrowth, and contained a mixture of Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum and Bacillus subtilis. Diet samples were plated in agar plates to quantify probiotic colony formation. Body weights of piglets were recorded before the beginning of the study and thereafter at 3-d intervals until the end of the study. On week 5 and 10, blood was collected 2-h post feeding via jugular venipuncture for analysis of liver biochemistry parameters. On week 10, all animals were euthanized and hepatic tissue was collected for histopathological analysis. In addition, jejunum, ileum, colon, cecum and rectum contents were collected for detection of probiotic DNA by PCR. CONP and HFFP diets increased (P < 0.01) number of colonies formed in agar plates compared to CON and HFF. CONP increased (P < 0.05) body weight compared to CON, HFF and HFFP. Serum liver enzymes, which are markers of hepatocellular degeneration, were higher (P < 0.001) in both HFF and HFFP compared to CON and CONP, and were not affected by probiotic supplementation. DNA of all probiotics except Lactobacillus plantarum was identified in the gut. Histopathological analysis of liver tissue showed extensive macrovesicular steatosis, hepatocellular degeneration, inflammation and necrosis in HFF and HFFP, but not in CON and CONP animals. In conclusion, 10-week probiotic supplementation does not ameliorate hepatic steatosis, degeneration and necrosis induced by high fructose high fat diet in neonate pigs. ARI#58873
Poster #: 224
Campus: CSU Chico
Poster Category: Disease (Pathogens)
Keywords: avian influenza virus, waterfowl,
Project Title: Isolation and Characterization of Avian Influenza Viruses in Northern California
Author List:
Bianchini, Elizabeth; Graduate, Biological Sciences, California State University, Chico, Presenting Author
Burris, Jessica; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author
Bogiatto, Raymond ; Faculty, Biological Sciences, California State University, Chico
Donatello, Robin; Faculty, Mathematics and Statistics, California State University, Chico
Cline, Troy; Faculty, Biological Sciences, California State University, Chico
Abstract: Recent human infections with influenza viruses of avian origin highlight the need for continued surveillance of avian influenza viruses (AIV) in waterfowl. In 2014, highly pathogenic avian influenza (HPAI) H5N8 first detected in South Korea entered North America through the Pacific Flyway, a major migratory route for waterfowl, and caused outbreaks in poultry in the United States. California’s Sacramento Valley is an important wintering site for waterfowl from which avian influenza viruses may be isolated. To better understand the risk posed by avian influenza viruses circulating in California, we collected cloacal swabs from 2,066 hunter-killed ducks across three hunting seasons at different locations in the Sacramento Valley. Sixteen waterfowl species were represented in our sampling. The presence of influenza viruses in cloacal swabs was determined by PCR for the matrix gene. The overall prevalence rate was 10.5% with diverse HA and NA subtypes represented. We observed a significantly higher positive rate in 2015-2016 (19.9%), a phenomenon that may be related to overcrowding on wetlands due to drought conditions. Northern shovelers had a statistically higher carriage rate (21.3%) relative to other species. Viral subtypes that were successfully isolated included one H9N2 virus and three H7N3 viruses. These viruses are of particular interest because these hemagglutinin (HA) subtypes have been reported to cause human infections in the past. Full genome sequencing revealed several genetic markers of mammalian adaptation. To test the hypothesis that these four AIV isolates are capable of replicating in mammalian cells and causing disease in mammals, they were characterized with respect to in vitro replication kinetics and their ability to cause disease in a mouse model of infection. All four viruses were capable of replication in mammalian cell culture at levels similar to a human H1N1 virus.
Poster #: 225
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: Caenorhabditis elegans, Candida , antifungal
Project Title: An Inexpensive Quantitative Method for Testing Anti-Fungal Drug Activity Using the Invertebrate Caenorhabditis elegans
Author List:
Pomytkina, Ielyzaveta; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Adler-Moore, Jill; Faculty, Biological Sciences, California State Polytechnic University, Pomona
Abstract: Introduction: Due to ethical and budgetary concerns associated with the use of vertebrate animals in research, interest in alternative models has increased over the past several decades. In the present study, we developed a Candida albicans quantitative infection model in Caenorhabditis elegans, a non-parasitic invertebrate nematode, to test the antifungal effects of liposomal amphotericin B (L-AmB).
Methods: To establish a lethal C. albicans infection, larval stage 4 worms [n=30/group (gp)] were fed various doses of yeast (2.5x10ex2 – 2.5x10ex5 cells) for 4h at 30°C. The infection was evaluated by monitoring worms for mortality and determining fungal burden in worm homogenates by plating for colony forming units every 24h for 4d post-challenge. To examine the worm’s ability to ingest L-AmB and to determine drug toxicity, uninfected worms were fed L-AmB (6.3 – 25 ug/gp) for 4h at 30°C, and drug toxicity evaluated by survival with drug concentrations determined by bioassay of worm homogenates. Lack of toxicity allowed us to evaluate L-AmB activity in worms challenged with 2.5x10ex5 yeast cells/gp and then treated with L-AmB (0.5 – 25 ug/gp), with survival rate and fungal burden to assess L-AmB treatment.
Results: C. albicans infection was established in worms challenged with all yeast doses, with optimum infection observed with 2.5x10ex5 yeast cells/gp (13% survival in infected worms vs. 87% in uninfected worms). We observed that uninfected worms could take up L-AmB at doses of 6.3 – 25 ug/gp and yet was not toxic for the worms (93-95% survival). In worms exposed to yeast and treated with L-AmB, complete clearance of infection was achieved with the higher doses (6.3 to 25 ug/gp), while lower doses (1.6 to 3.1 ug/gp) significantly reduced the fungal burden (p≤0.05). Infected worms, not treated with L-AmB had only 10% survival, while L-AmB improved survival in a dose-dependent manner giving 40% survival for 0.5 ug L-AmB/gp and 100% survival for doses of 6.3 ug/gp and higher.
Conclusion: By using fungal burden as a readout of efficacy, along with survival, we have established a quantitative, reproducible, flexible method for examining the activity of L-AmB in C. elegans which could be expanded for use in evaluating other antifungal drugs and different pathogenic fungi.
Poster #: 226
Campus: CSU Northridge
Poster Category: Disease (Pathogens)
Keywords: Epidemiology, Antibiotic Resistance, Agriculture
Project Title: Distribution, Characterization, and Whole Genome Sequencing of Carbapenem-Resistant Bacteria in Los Angeles Area Soils
Author List:
Lopez, Nicolas; Undergraduate, Biology, California State University, Northridge, Presenting Author
Farsar, Cameron
Ruiz Rueda, Cristian; Faculty, Biology, California State University, Northridge
Abstract: Infections caused by Carbapenem-resistant bacteria (CRB) are hard to treat because of their resistance to carbapenems, a class of “last-resort” beta-lactam antibiotics. Because carbapenems are only used in health care facilities the distribution and evolution of CRB has rarely been studied outside these settings. Our goal is to investigate the distribution and frequency of these bacteria in the environment. We also want to know genetic and biochemical properties of significant isolates. There have been studies on CRB in natural soil in Algeria, England and in agricultural soils of Texas and New Mexico. However, there have not been any studies on CRB in soil on the West Coast of the United States. In this project we surveyed eight soil samples in the Western San Fernando Valley and East Ventura County for CRB. We used selective media with meropenem to isolate CRB, and used 16S rDNA PCR, sequencing, and NCBI-BLAST to identify them. Then we used antibiotic succeptibility tests and biochemical assays to determine the antibiotic resistance profile. We have found several carbapenem-resistant isolates of the Stenotrophomonas, Pseudomonas, Cupriavidus, Staphylococcus, Bacillus, and Achromobacter genera, which are all opportunistic pathogens. Antibiotic succeptibility tests showed that all of the isolates were resistant to different carbapenems and other classes of antibiotics. Whole Genome Sequencing analysis of one of these isolates, Pseudomonas alkalphenolica, revealed that it carries two different beta-lactamase genes. One of the genes is similar to blaLCR-1, a Class-D beta-lactamase gene, related to the blaOXA-48 gene, encodes for a carbapenem-degrading enzyme found in many clinical carbapenem-resistant isolates. The other gene is similar to blaAMP-C, a Class-C beta-lactamase. Further experiments are in progress to clone and characterize both beta-lactamase genes. These findings indicate that soil is a reservoir for CRB in the Los Angeles area. Carbapenem resistance could be natural in soil bacteria. High rates of carbapenem resistance could be a result of the use of beta-lactams such as penicillin and its derivatives in agriculture. Most of our isolates are opportunistic pathogens that may end up in clinical settings or serve as antibiotic-resistance determinant donors.
Poster #: 227
Campus: CSU Fresno
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: development, mitochondria, genetics
Project Title: Mitochondrial-nuclear genetic incompatibility causes hybrid developmental delay
Author List:
Takhar, Sukhmeet; Undergraduate, Biology, California State University, Fresno, Presenting Author
Ross, Joseph; Faculty, Biology, California State University, Fresno
Abstract: To understand how genetics impacts development, we use the model organism Caenorhabditis briggsae. This microscopic worm has a short generation time and produces many offspring, which allows us to study development of a large sample of individuals. Previous research indicates that mitochondrial and nuclear genotypes, and their interactions, might affect C. briggsae hybrids by reducing fitness, particularly by delaying development. Thus, we investigated the growth rates of cytoplasmic-nuclear hybrids (cybrids), which contain the mitochondrial genome of one parent strain and the nuclear genome of the other. At regular intervals during larval development, we collected micrographs of multiple individuals from replicate cybrid lines as well as from the parent populations. We then determined the development rates of the strains by measuring the total length of each individual at each time point. Cybrid strain development was significantly delayed when compared to the parent strains. Each of the strains also developed at a different rate when compared with each other. Because the cybrid strains only genetically differ from the parents strains by their mitochondrial genotypes, these data show that disrupting mitochondrial and nuclear genetic interactions impacts organismal fitness. Future efforts will seek to identify the specific mitochondrial and nuclear genetic variants that are involved in this interaction. Ultimately, this will help us to understand the molecular and cellular mechanisms that become disrupted to elicit the cellular- and organismal-level phenotype of developmental delay.
Poster #: 228
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: gene regulation, promoter, bioinformatics
Project Title: Characterizing the Promoter Region of Recombination Signal Binding Protein for Immunoglobulin Kappa J region
Author List:
Salazar, Jonathan; Graduate, Biology, California State University, Northridge, Presenting Author
Jocol-Perez, Lesslie; Staff, Biology, California State University, Northridge, Presenting Author
Malone, Cindy; Faculty, Biology, California State University, Northridge
Abstract: Recombination Signal Binding Protein for Immunoglobulin Kappa J region (RBPJ) is a protein involved in the transcriptional regulation of the Notch signaling pathway. RBPJ protein can act as either a repressor or activator depending on whether it is bound to a Notch intracellular domain. The significance of RBPJ lies in its ability to regulate the Notch signaling pathway that is vital in cancer cell proliferation. Since we understand that transcription factor proteins have regulatory effects when bound to the promoter regions of genes we use bioinformatic techniques to help in determining which regions of the RBPJ promoter are highly conserved. The focus of this project is to characterize the proximal promoter region of the RBPJ promoter and inform on regions that have an observable impact on gene expression levels. To achieve this goal, we genetically transformed chemically competent Escherichia coli cells with a luciferase expression vector containing the promoter region of RBPJ. To inform on shorter segments of the RBPJ promoter we used an exclusion PCR method which allows us to remove fragments of the promoter and more easily characterize fragments. The recombinant plasmids containing the RBPJ promoter with different regions of it removed are then transfected into Human Embryonic Kidney Cells (HEK 293T) using an Effectene Transfection method. The luciferase proteins are extracted from the cells using a passive lysis method. A luciferase assay is performed on all the protein lysates to determine the regulatory effect different regions of the RBPJ promoter have on luciferase expression. We saw varying levels of expression between 3 of our RBPJ promoter constructs. The assay informed that there is at least 1 repressor in the region between the -750 and -483 RBPJ deletion construct and that there is at least 1 activator between the -483 and -200 deletion constructs. To determine which regions have the highest impact on expression we will be knocking out short DNA regions, ranging from 10-20 base pairs in length, that were chosen based off potential transcription factor binding sites that were derived using bioinformatic techniques. Determining which transcription factor is having the strongest effect on gene expression will be useful for future potential therapies involving the knockdown or overexpression of RBPJ which is a key transcriptional effector in the Notch signaling pathway which is an essential pathway involved in cell proliferation.
Poster #: 229
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Sinorhizobium meliloti, Microbiology, site-directed mutagenesis
Project Title: Testing the role of a consensus sequence for ChvI-dependent gene regulation in the nitrogen-fixing bacterium Sinorhizobium meliloti
Author List:
Ortega, Jesus; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Dela Cruz, Francesca; Undergraduate, Biological Science, California State University, Fullerton
Chen, Esther; Faculty, Biological Science, California State University, Fullerton
Abstract: Sinorhizobium meliloti forms an endosymbiotic relationship with legume plant hosts, including Medicago sativa. The symbiosis provides plants with biologically accessible nitrogen, which has an important role in determining agricultural productivity. The conserved ExoS/ChvI two-component signaling pathway is necessary for the transition between the symbiotic and free-living states of S. meliloti, and controls expression of genes involved in exopolysaccharide production, motility, and nutrient utilization. After phosphorylation by the ExoS histidine kinase, ChvI binds to promoter regions and regulates gene expression. Previous work in our laboratory identified a 15-bp consensus sequence upstream of ChvI transcriptional target genes; this motif was hypothesized to be important for gene regulation by ChvI. We tested the importance of this motif for ChvI-dependent gene regulation by using site-directed mutagenesis to make substitution or deletion mutations in the motifs upstream of the exoY and SMc00084 genes. The upstream regions of both genes harboring the mutated motifs were fused to the reporter gene encoding beta-glucuronidase (GUS), and these transcriptional fusions were introduced into wild-type S. meliloti. We compared GUS activity of S. meliloti strains harboring the wild-type, deletion mutation, and substitution mutation motifs. For both genes, we observed a large decrease in GUS activity in the strains with the substitution or deletion mutations in the motif compared to strains with the wild-type motif. Therefore, we conclude that the 15-bp consensus sequence is important for regulation of the exoY and SMc00084 genes that are transcriptionally controlled by ExoS/ChvI. This work was supported by the Cal State Fullerton MARC Program grant funded by NIH [2T34GM008612-23] and by NSF IOS-0818981 to E.J.C.
Poster #: 230
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: microRNA, ICER, insulin
Project Title: cAMP Pathway Dependent Mechanism Stimulates Insulin Secretion via Regulation of miR-375 and ICER Gene Expression
Author List:
Nielsen, Ryan; Graduate, Biology, California State University, Chico, Presenting Author
Frias, Stephanye ; Graduate, Biology, California State University, Chico, Presenting Author
Teazis, Jonathan; Undergraduate, Biology, California State University, Chico
Keller, David; Faculty, Biology, California State University, Chico
Abstract: Type 2 diabetes (TD2) is an increasing condition in our society and treatments are being heavily pursued, targeting potential environmental and genetic pressures. The area of study for this project is a microRNA called miR-375 which is overexpressed in people with TD2 and has been linked to decreased insulin secretion and beta cell proliferation. Investigation into the transcription factor inducible cAMP early repressor (ICER) as an intermediate regulator of miR-375 was proposed because both were found to be regulated by cAMP. To study this interaction, mRNA from rat INS-1 cells and human islets were isolated to conduct 3’ rapid amplification of cDNA ends (RACE). An adapter primer is used to target the poly(A) tail region and try to capture the unknown 3’-mRNA sequence of ICER that lies between the exon and poly(A) tail. We predict that miR-375 binds to ICER at the 3’ end, which makes locating ICER the first step to understanding how miR-375 regulates insulin secretion. Secondly, an insulin enzyme-linked immunosorbent assay (ELISA) was performed on INS-1 cells overexpressing a miR-375 mimic or a control microRNA in different glucose concentrations, to see if insulin secretion rates varied. Results showed that when miR-375 was overexpressed, insulin secretion rates were higher than with a control in either glucose concentration. Insulin ELISAs were performed on INS-1 cells, and human islets with a miR-375 inhibitor, and a negative inhibitor to see if insulin secretion rates would vary in different concentrations of glucose, as well as in species specific cell lines. Next, Icer’s expression was measured in human embryonic kidney cells (HEK-293T) when exposed to the cAMP stimulating drug forskolin or when co-transfected with a plasmid containing ICER and an ɑ-ICER small interfering RNA (siRNA) using quantitative real time PCR. A primer set targeted against an exon found in all Icer transcript variants was used to measure Icer expression in treated cells. Icer’s expression was increased in cells treated with forskolin while Icer’s expression was knocked down when co-transfected with ɑ-ICER siRNA. We confirmed a decrease in insulin secretion when introduced to miR-375 and investigated the interaction of ICER and miR-375 binding at the 3’ end. Additionally, cAMP is heavily pursued as a treatment for TD2 symptoms so the determination that both ICER and mir-375 are regulated by this pathway provides evidence for a cAMP dependent mechanism for gene expression.
Poster #: 231
Campus: San Francisco State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: rhizobium, symbiosis, bacterial genetics
Project Title: A protease and a lipoprotein jointly regulate microbe-plant symbiosis
Author List:
Ceron, Josue; Undergraduate, Biology, San Francisco State University, Presenting Author
Chen, Joseph; Faculty, Biology, San Francisco State University
Abstract: Sinorhizobium meliloti serves as a model alpha-proteobacterium for investigating microbe-host interactions, in particular rhizobium-legume symbiosis that enables nitrogen fixation. To achieve symbiosis, the rhizobium and a compatible host must exchange a complex series of chemical signals, including exopolysaccharide-I (EPS-I, also known as succinoglycan), a bacterial product that contributes to successful infection of plant roots. In S. meliloti EPS-I production is controlled by the conserved ExoS-ChvI two-component signal transduction pathway. Periplasmic ExoR associates with the ExoS histidine kinase and negatively regulates ChvI-dependent expression of exo genes, necessary for EPS-I synthesis. We show that two previously uncharacterized proteins, LppA (a lipoprotein) and JspA (an extracytoplasmic metalloprotease), jointly influence EPS-I synthesis by modulating the ExoR-ExoS-ChvI pathway. Deletions of jspA and lppA led to reduced EPS-I levels, as evaluated via calcofluor fluorescence, and competitive disadvantage during host colonization. Transcriptional fusion reporter assays demonstrated that JspA and LppA require each other to positively regulate expression of representative exo genes. Microarray analysis further revealed that JspA affects expression of numerous genes in the ChvI regulon, including chvI itself. Epistasis analysis indicated that JspA acts upstream of the signaling pathway: (1) while the exoR::Tn5 mutant exhibited elevated EPS-I levels and the jspA mutant reduced EPS-I levels, the double mutant overproduced EPS-I; and (2) increased expression of exo genes when jspA is overexpressed requires ChvI activity. Finally, overexpression of JspA caused a slight increase in exoR transcription but reduced steady-state levels of the ExoR protein, suggesting that JspA contributes to ExoR degradation. Akin to signaling pathways that sense extracytoplasmic stress in other bacteria, JspA and LppA may monitor periplasmic conditions during interaction with the plant host to adjust accordingly expression of genes that contribute to efficient symbiosis. By investigating the molecular mechanisms underlying host colonization in our model system, we expect to uncover the roles that conserved signaling molecules play during infection in related alpha-proteobacteria.
Poster #: 232
Campus: CSU San Bernardino
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: influenza virus , RNA expression , protein interaction
Project Title: Characterize Influenza NP-NS1 interaction as novel antiviral target
Author List:
Gazca , Cinthia E. ; Undergraduate, Biology , California State University, San Bernardino, Presenting Author, Nagel Award Finalist
Newcomb, Laura L; Faculty, Biology , California State University, San Bernardino
Abstract: Influenza virus is an economic and global health concern. The virus rapidly evolves to evade vaccine induced immunity and gain resistance to antiviral therapies. Yearly vaccination is required to control seasonal influenza, while pandemic prevention relies on effective antivirals. The viral ribonucleoprotein (vRNP) is responsible for influenza RNA synthesis and is comprised of many nucleoproteins (NP), the RNA genome segment, and the RNA dependent RNA polymerase (RdRP). NP is a highly conserved structural component of the vRNP, but also interacts with viral and host factors to regulate viral RNA expression, making NP interactions of interest as targets for influenza antivirals. Influenza nonstructural protein 1 (NS1) is primarily an interferon inhibitor, but has multiple functions and is reported to interact with NP. Our aim is to establish if NS1 protein enhances viral RNA expression through interaction with NP, making this protein interaction a novel antiviral target. To accomplish our aim, reconstituted vRNPs were expressed in the presence or absence of NS1 in human embryonic kidney cells (293T), with an intact interferon response, and green monkey kidney cells (VERO), lacking the antiviral interferon response. M-GFP-vRNA was used as template in reconstituted vRNPs to assess vRNP function indirectly by GFP expression. Total RNA was isolated to confirm changes in gene expression at the RNA level. RNA concentration was determined by optical density, and equal concentrations resolved on a 1% agarose gel to confirm RNA integrity. RNA was reverse transcribed with oligo dT and quantitative PCR performed with specific primers. Our results show addition of NS1 enhances both protein and RNA expression from reconstituted vRNPs in 293T cells. This enhancement is even more observable at the protein level in VERO cells, emphasizing this observation is interferon independent. RNA expression in VERO cells is being analyzed and expected to confirm changes in gene expression occur at the RNA level. To evaluate NP-NS1 interaction, 293T cells were transfected to express WT-NP with or without NS1. Total protein extracts were isolated and NP complexes immunopurified and examined via western blot to confirm NP-NS1 interaction. We will examine both vRNP activity and NP interaction with NS1 mutants: NS1 R38A (RNA binding deficient), NS1 G184R and NS1 W187R (CPSF30 binding deficient). Our experiments will characterize NP-NS1 interaction and NS1 impact on vRNP activity.
Poster #: 233
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: RNA-seq, Polerovirus, Nicotiana glutinosa
Project Title: Differential expression of Nicotiana glutinosa genes in defense against Polerovirus infection
Author List:
Weir, Allyson; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author, Nagel Award Finalist
Der, Joshua; Faculty, Biological Sicence, California State University, Fullerton
Sacco, Melanie; Faculty, Biological Science, California State University, Fullerton
Abstract: Plants defend themselves against infectious pathogens through inducible defenses that operate at the molecular level through immune receptors that are activated by pathogen elicitors. Two inducible responses to pathogens are termed extreme resistance, in which the pathogen is eliminated without cell death, and the hypersensitive response, which limits the pathogen to the infected area by local programmed cell death. When infected with different members of the Polerovirus genus, different genetic backgrounds of Nicotiana glutinosa display either hypersensitive response or extreme resistance. N. glutinosa accession TW59 exhibits hypersensitive response when infected with Turnip yellows virus (TuYV) as well as Potato leaf roll virus (PLRV), while accession TW61 exhibits hypersensitive response only when infected by PLRV and exhibits extreme resistance when infected by TuYV. To determine the gene expression changes that may explain the different outcomes observed, RNA was isolated from virus-challenged and control plants at 36 hours post infection, a time point just before the visible onset of cell death, from control leaves and leaves infected by TuYV or PLRV from the two accessions for analysis by Next Generation RNA-sequencing. We assembled 39,151 and 37,693 contigs in TW59 and TW61, respectively, of which 15,970 contigs and 484 contigs were significantly differentially expressed in the TW59 and TW61 transcriptomes, respectively. The Argonaute-1 protein is known to be down-regulated by Poleroviruses; however, the transcript level was not significantly altered in either transcriptome, providing evidence that it is down-regulated at the protein level only. Fifteen contigs from the TW61 transcriptome were selected as gene candidates for validation using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Four candidates were only differentially expressed in PLRV-infected samples, four candidates were only differentially expressed in TuYV-infected samples, and seven candidates were differentially expressed when infected by both viruses. We hypothesize that candidate genes exclusively differentially expressed in PLRV-infected plants will be associated with the manifestation of cell death, and that candidate genes specific to TuYV infection may function in extreme resistance, with potential roles in the execution phases of these inducible defense responses.
Poster #: 234
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: lipid droplet, phagocytosis, Drosophila
Project Title: Lipid Droplet Dynamics during Phagocytosis in Drosophila Hemocytes
Author List:
Myers, Amber; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Ogundipe, Eniola; Undergraduate, Biological Science, California State University, Fullerton
Brennan, Catherine; Faculty, Biological Science, California State University, Fullerton
Abstract: Phagocytosis is an immune defense in which specialized white blood cells, such as the mammalian macrophage and Drosophila hemocyte, engulf and kill extracellular microbes into a vesicle called the phagosome. Although the mechanisms used to kill microbes in the phagosome are well known, such as production of reactive oxygen species, it remains unclear how these killing mechanisms are regulated. Pathogenic Mycobacteria have virulence factors that subvert killing in the phagosome. In order to understand the interactions that occur between the phagocyte and pathogen, we must first know how phagocytosis is regulated. We are interested in the lipid droplet (LD) organelle and its role during phagocytosis. LDs store neutral lipids such as triacylglycerides and regulate lipid metabolism. When pathogenic Mycobacteria are engulfed, LDs accumulate in the hemocyte and mammalian macrophage and localize to the Mycobacteria-containing phagosome, where they are thought to serve as a nutrient source for Mycobacteria, thus promoting bacterial survival within the phagosome. However, the role of LDs in normal phagosome function, where the bacterium does not manipulate killing mechanisms, is not well known. Our aim is first to describe LD behavior during phagocytosis of non-pathogenic bacteria, including LD abundance and localization over time. Using fluorescent microscopy, we found that LDs accumulate in hemocytes up to three hours following the uptake of non-pathogenic bacteria and declined thereafter. Interestingly, the LDs did not localize to the phagosome, but were dispersed through the cell. These results suggest LD accumulation is a normal phagocytic response and LDs do not typically associate with phagosomes. Our second aim was to identify the source of lipids to form LDs and determine whether hemocytes synthesize lipids themselves or scavenge them from the extracellular environment. To test the latter possibility, we conducted ex vivo phagocytosis experiments where LD accumulation is slightly impaired using media containing various lipid content. We found increasing lipid content did not restore LD accumulation during ex vivo phagocytosis. Our results suggest hemocytes synthesize lipids rather than scavenge lipids from the extracellular fluid. In future studies, we hope to understand the contributions of LDs in phagocytosis and; anticipate they play a protective role against oxidative stress. We would like to acknowledge CSUPERB and NIH for supporting this work.
Poster #: 235
Campus: Humboldt State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Rickettsia, Complementation, Cloning
Project Title: Complementation of the folA and folC deletion in Escherichia coli by Rickettsia genes encoding dihydrofolate reductase and dihydrofolate synthase
Author List:
Schafer, Daniel; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
Hill, Brandon; Graduate, Biological Sciences, Humboldt State University, Presenting Author
Shrotri, Rohan; Undergraduate, Biological Sciences, Humboldt State University
Zhong, Jianmin; Faculty, Biological Sciences, Humboldt State University, Presenting Author
Abstract: Tick-borne diseases pose an imminent threat as climate change has fostered broadening of tick-borne disease (TBD) vector habitats, increasing tick populations, and projected increases in disease rates. In searching for TBD vector control targets, our lab discovered an endosymbiotic bacterium capable of de novo folate biosynthesis, Rickettsia Endosymbiont Ixodes pacificus (REIP), that is present, and 100% maternally transmitted, in TBD vector Ixodes pacificus. Our goal is to validate the posited nutritive mutualism between I. pacificus and REIP, which could then be exploited in a TBD vector control program. Our lab has initiated two complementation assays in parallel to demonstrate the functions of two rickettsial biosynthesis genes, the dihydrofolate reductase-encoding gene folA and dihydrofolate synthase-encoding gene folC, which will elucidate the hypothesized folate provisioning role of REIP within its tick host. To overcome the toxicity issue of folate gene products, we cloned folA and folC genes of REIP into a custom-made low-copy number plasmid, TransBac. To facilitate the cloning, we first cloned the two genes of REIP in the pSC-A-amp/kan plasmid using StrataClone PCR Cloning Kit. Utilizing SfIl and AvrII restriction enzymes, double digestion removed either folA or folC gene of REIP from the pSC-A-amp/kan plasmid. The TransBac plasmid, containing a tetracycline resistance gene, was used as the recipient vector in the subcloning process. Transformation of the ligation products into 10G chemically competent E. coli was followed by tetracycline selection on LB plates. Colony PCR was then successfully applied to screen transformants for the presence of rickettsial folA and folC genes. Visualization of the PCR products on an agarose gel confirmed the sizes of DNA fragments akin to the amplified loci containing the folA and folC genes. Plasmid DNA was isolated from transformed E. coli and sequencing analysis is pending. A final PCR amplification using the purified clone DNA provided additional verification of the constructions of the clones. Complementation assay of mutant E. coli containing folA and folC knockouts will determine if the rickettsial folA and folC genes will restore wild type function and maintain viability in a folate deprived environment. Our successful constructions of folA and folC clones of REIP is a crucial step in demonstrating the functional homology between rickettsial and E. coli folate biosynthesis genes.
Poster #: 236
Campus: CSU Sacramento
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: gene amplification, Acinetobacter, genetics
Project Title: Enrichment of precursor cells of gene amplification mutants prior to selective pressure in Acinetobacter baylyi
Author List:
Herrmann, Jennifer; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Difuntorum, Shyla; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Quiñones-Soto, Semarhy; Faculty, Biological Sciences, California State University, Sacramento
Reams, Andrew; Faculty, Biological Sciences, California State University, Sacramento
Abstract: Gene amplification mutations are strong contributors to the formation of cancer cells and building resistance to antibiotics in many pathogens. Recent studies suggest these important mutations are induced by stress imposed during growth-limiting environments. In contrast, an alternative theory suggests these mutations are present prior to the selective environment and provide fitness advantage after selection is imposed. In this study, we test both theories using a model system in the genetically tractable bacteria Acinetobacter baylyi, where gene amplification mutants are exclusively selected and continually accumulate under prolonged growth-limiting selective conditions in benzoate minimum media. Here, replica-printing experiments were performed to determine whether the gene amplification mutants arise either prior or during the selective-stress exposure. For these experiments, approximately 10,000 colonies of the parent strain were grown on non-selective rich media agar plates, where gene amplification is not required for growth. These non-selective plates were incubated at 30C for 16-18 days then replica-printed onto a non-selective rich media master plate and three selective benzoate agar plates. Our results show gene amplification mutant colonies continually arose on the selective benzoate agar plates, increasing in number over several days of incubation. Interestingly, these mutant colonies arose in the same positions on the three benzoate replica-plates, strongly suggesting the mutants existed before selection was imposed. The corresponding position of the three sibling mutant colonies was traced back to the location on the non-selective master plate to collect the original cells. These enriched cells were resuspended, serial-diluted, and plated onto non-selective agar plates to get approximately 50,000, 5,000, 500, and 50 colonies per plate. After 7 days, the plates were replica-printed onto one non-selective and three benzoate agar plates. Our results show the majority of these purified colonies on nonselective plates grew in the same positions on the benzoate replica-plates. Isolated colonies were found to contain the precursors to gene amplification, such as duplications or triplications. This research was supported by the CSUPERB New Investigator Award, the CSUS Summer Undergraduate Research Experience (SURE) Award, and the NIGMS-RISE under a grant from the NIH (1R25GM122667).
Poster #: 237
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Pancreatic Cancer, Microenvironment Remodeling, Cytoskeleton Proteins
Project Title: Functional studies of Retinoic Acid Induced 14 during microenvironment remodeling and progression in pancreatic cancer
Author List:
Runa, Farhana; Postdoc, Biology, California State University, Northridge
Gonzalez, Carolina; Undergraduate, Biology, California State University, Northridge, Presenting Author
Cox, Nathan; Duke University
Adamian, Yvess; Undergraduate, Biology, California State University, Northridge
Hong, Julie; Undergraduate, Biology, California State University, Northridge
Bhakta, Kishan; Undergraduate; University of Utah, Biology, California State University, Northridge
Hoover, Malachia; Undergraduate, Biology, California State University, Northridge
Boyce, Michael; Duke University
Kelber, Jonathan; Faculty, Biology, California State University, Northridge
Abstract: The extensive desmoplastic response in the pancreatic ductal adenocarcinoma (PDAC) microenvironment is one of the primary hurdles toward developing effective therapies to combat this deadly malignancy. Since O-GlcNAc modification of SEC23A mediates proper trafficking of collagen (a primary constituent of the PDAC microenvironment), we reasoned that SEC23A interacting partners, which are upregulated in PDAC, may yield novel strategies for targeting the PDAC stroma. In this regard, we identified RAI14 (Retinoic Acid Induced 14 or Ankycorbin or NORPEG) as a candidate SEC23A binding partner using GlcNDAz-mediated crosslinking and mass spectrometry, and ongoing work aims to confirm/validate these preliminary biochemical results. Notably, both RAI14 and SEC23A are among the top 6% of genes that are significantly upregulated in pancreatic cancer tissue relative to normal pancreatic tissue. Further, we report that RAI14 co-localizes with actin stress fibers in PDAC cells and that RAI14 knockdown reduces PDAC cell migration and proliferation/survival. Using the CRISPR-CAS9 system, RAI14 was depleted from PDAC as well as chondrosarcoma and non-malignant kidney epithelial cells to further test for collagen expression/secretion, chemotherapy responsiveness and tumor growth/metastasis. The long-term goal of these studies is to elucidate the mechanistic basis for PDAC progression and therapy resistance. Acknowledgments and Funding Agency: Northridge (CSUN) College of Mathematics and Science, the Sidney Stern Memorial Trust, the CSU Program for Education and Research in Biotechnology (CSUPERB), Medtronic, and the Building Infrastructure Leading to Diversity (BUILD) Promoting Opportunities for Diversity in Education and Research (PODER), NIH-NIGMS and Dr. Gary and Vera Sutter.
Poster #: 238
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: t brucei, parasite, ion channel
Project Title: Localization and role of a mechanosensitive channel in the procyclic form of Trypanosoma brucei.
Author List:
Hernandez, Monica; Graduate, Biology, California State University, Fullerton, Presenting Author
Nguyen, Kristy; Undergraduate, Biology, California State University, Fullerton
Dave, Noopur; Graduate, Biology, California State University, Fullerton
Jimenez-Ortiz, Veronica; Faculty, Biology, California State University, Fullerton
Abstract: Trypanosoma brucei (T. brucei) is a protozoan parasite transmitted by the tsetse fly and the causative agent of African Sleeping Sickness. Once the parasite has established infection in the host it can pass the blood brain barrier causing psychological and neurological disturbances. At this stage, the mortality rate is close to 100%. Current treatment causes severe side effects and often require prolonged intravenous administration, highlighting the need for new therapeutic options. Potential targets for drug development include mechanosensitive channels (MSCs) which have been shown to contribute to bacterial virulency. Screening of T. brucei’s genome revealed a putative mechanosensitive channel, TbMscS. TbMscS shares 64% identity with a recently characterized MscS-like channel in T. cruzi and 31% identity with E. coli MscS. The protein has a predicted topology and gating mechanisms similar to the one described in E. coli. We have evaluated the localization of the channel in bloodstream and procyclic forms of the parasite using specific antibodies. Our results indicate it is distributed along the flagellar attachment zone and the flagellar pocket. Knockdown of the channel by RNAi achieved more than 80% efficiency allowing for the phenotypic characterization of the cell line. Knockdown parasites show similar growth compared with the controls but there is an increase number of cell with abnormal morphology. Osmotic stress experiments suggest the channel may play a role in stress responses upon hyper-osmotic shock. Our results indicate that TbMscS is a mechanosensitive channel important for regulating cell division and osmotic compensation in the procyclic form of T. brucei. Since bacterial-like MscS channels are not present in humans, TbMscS could be a promising drug target against an economic and medical burdensome disease.
Poster #: 239
Campus: CSU Long Beach
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Ovary, Reproduction, Hormone
Project Title: Short photoperiod reduces RNA binding protein Mex3a; however, does not alter expression of endoribonuclease Dicer1 in ovaries of Siberian hamsters (Ph
Author List:
Song, Wei; Undergraduate, Department of Biological Sciences, California State University, Long Beach, Presenting Author
Chin, Catherine H.; Undergraduate, Department of Biological Sciences, California State University, Long Beach, Presenting Author
Young, Kelly A.; Faculty, Department of Biological Sciences, California State University, Long Beach
Abstract: Follicle stimulating hormone (FSH) stimulates ovarian function during the breeding season in many seasonally-breeding vertebrates. Exposure of female Siberian hamsters (Phodopus sungorus) to short day (SD) lengths typical of the non-breeding season reduces plasma FSH, which decreases follicle development and ovulation. This photoperiod-induced regression of ovarian function is reversed with subsequent exposure to stimulatory long days (LD). While FSH is critical, recent studies have also implicated the importance of post transcriptional RNA modification as well as the actions of small non-coding RNAs called microRNAs (miRNAs). Dicer1, an endoribonuclease necessary for the production of miRNAs involved in the regulation of oogenesis, is essential for the normal maturation of oocytes. Mex3a, an RNA-binding protein important in post-translational modification, may be involved in cell proliferation and differentiation, key aspects of ovarian follicle development. Most studies investigating RNA binding and modification have focused on cycling ovaries and have not examined the interaction with FSH. Therefore, we hypothesized that Dicer1 and Mex3a would be: 1) present in ovaries of Siberian hamsters and 2) differentially regulated across photoperiod and presence/absence of FSH. Hamsters (n=60) were exposed to either: LD to promote cyclicity, SD to induce regression, or were transferred from SD to LD to stimulate the return to ovarian function (post transfer, PT). The group of PT females was subdivided into additional groups: no treatment (PT), mannitol vehicle treatment (PTV), treatment with acyline to reduce FSH (PTA), and treatment with both acyline and FSH (PTAF). Dicer1 mRNA was present in all groups with no differences between different photoperiod exposures or treatments (p>0.05). Mex3a mRNA was present in LD control ovaries, and significantly declined in SD ovaries (p<0.05). Although Mex3a mRNA levels did not differ between the LD and PT groups (p>0.05); Mex3a mRNA was significantly lower in the acyline-treated PTA females as compared to LD controls (p<0.05). FSH treatment did not restore Mex3a levels; they remained reduced in PTAF as compared to LD values (p<0.05). While Mex3a function peaked in fully functional ovaries, Dicer1 was expressed regardless of ovarian function. Neither factor appears to be directly regulated by FSH, suggesting that some aspects of seasonal change in ovarian function are independent of FSH. Supported by: NIH SCORE 1SC3GM116696.
Poster #: 240
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila, Notch, ethanol
Project Title: Dementin and Notch Implicate the Gamma Secretase Complex in the Developmental Response to Ethanol
Author List:
Darwish, Nahed; Graduate, Biological Sciences, San José State University, Presenting Author
Belhorma, Khaoula; Graduate, Biological Sciences, San José State University, Presenting Author
French, Rachael; Faculty, Biological Sciences, San José State University
Abstract: Every year 40,000 children are born with fetal alcohol syndrome disorder (FASD) as a result of maternal alcohol consumption during pregnancy. These children often go on to develop neurodegeneration similar to that observed in Alzheimers Disease (AD).
We have a well-established Drosophila melanogaster model for the study of FASD; in addition, Drosophila have been used as a model for the study of AD. Thus, flies are an excellent model organism to study the intersection between alcohol-induced developmental toxicity and AD-like neurodegeneration.
We previously identified several alleles of dementin (dmtn), the Drosophila ortholog of TMCC2, that convey sensitivity to developmental alcohol exposure. In addition, quantitative reverse-transcriptase mediated PCR (RT-qPCR) analysis demonstrates that dmtn transcript levels is decreased an average of 64% in larvae reared in ethanol, demonstrating that dmtn expression is disrupted by developmental ethanol exposure.
TMCC2 is a human protein that physically interacts with amyloid beta precursor protein (APP), a key protein in AD pathology, and mutations in dmtn cause both abnormal metabolism of the fly homolog of APP (APP-like, APPL) and neurodegeneration. However, little else is known about the function of this protein. In order to begin to characterize the role of dmnt in neurodegeneration and the developmental response to ethanol, we tested developmental ethanol sensitivity in flies mutant for genes known to participate in APPL metabolism.
One such gene, presenilin (psn), encodes a component of gamma secretase, a proteolytic enzyme that participates in the cleavage of many proteins, including APPL and Notch. Flies mutant for psn were sensitive to ethanol during development, displaying reduced survival similar to that seen in dmtn mutants (16 to 33% reduction in survival compared to wild type controls). In addition, we tested two heterozygous loss of function Notch alleles, and in both cases, we found reducing Notch to be protected against developmental ethanol toxicity. We will present these results, as well as the results of testing the roles of additional components of the gamma secretase complex and Notch pathway in the cellular response to developmental ethanol exposure.
Poster #: 241
Campus: San Diego State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Arabidopsis, Epigenetics, Heat Stress
Project Title: The Role of Epigenetics in Plant Adaptation Across an Elevational Cline in Arabidopsis thaliana
Author List:
Fabunan, Rudy; Graduate, Biology, San Diego State University, Presenting Author
Tonsor, Stephen; Carnegie Museum of Natural History
Waters, Elizabeth; Faculty, Biology, San Diego State University
Abstract: Plant communities are at increasing risk of extinction as temperatures continue to increase. Due to their sessile nature plant species must adapt to increasing heat stress in place. Previous studies of standing genetic variation and gene expression patterns have found evidence of adaptation to changing temperatures. However, the role of epigenetic variation in adaption is still unclear. This is despite the fact that plant methylation patterns are known to change in response to stress. The goal of this project is to evaluate the hypothesis that methylation patterns differ in heat tolerant and intolerant plants and that these differences play an important role in adaptation to abiotic stress. I have examined the patterns of genetic and epigenetic variation in 11 populations of A. thaliana, grouped into two different population levels based on elevation, that have been collected along an altitudinal and temperature cline from North East Spain. The methods used include bioinformatics pipelines to evaluate genetic variants such as single nucleotide polymorphisms (SNPs), methylome analysis of the two population groups, and gene ontology (GO) term analysis. In this study, I have found genetic variation between the high elevation and low elevation plants but, based on SNP effect predictions, most of these differences are not predicted to have major effects on protein function. However, methylation analysis did reveal that lowly and highly methylated genes are involved in different cellular processes. Lowly methylated genes are involved in response pathways, while highly methylated genes are involved in cellular organization and other multiple biological processes. This suggests that genes that are lowly methylated are responsible for fast acting response to abiotic stimulus and thus could be involved in adaptation.
Poster #: 242
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: CRISPR, Hematopoietic Stem Cells, Genome editing
Project Title: The Effect of Valproic Acid on CRISPR-Cas9-mediated genome editing
Author List:
Huang, Codey Y.; Undergraduate, Biological Sciences, San José State University, Presenting Author
Yamamoto, Emiko L.; Undergraduate, Biological Sciences, San José State University, Presenting Author
Johnston, Jennifer M.; Faculty, Biological Sciences, San José State University
Abstract: Genome editing utilizing the CRISPR-Cas9 system has become more prominent due to increased specificity and adaptability to multiple targets. However, homology directed repair (HDR), the process by which DNA templates are integrated back into the genome, is still inefficient with currently available technologies. Valproic acid (VPA), an FDA approved antiepileptic, can potentially improve HDR efficiency. As a histone deacetylase inhibitor, VPA prevents DNA from being wrapped tightly around histones. Since histone acetylation enhances DNA expression, prior treatment with VPA may increase DNA accessibility to the Cas9 endonuclease. In these preliminary experiments, we are testing this hypothesis in K562 cells, a myeloid-erythroid cell line which is representative of one half of the hematopoietic stem and progenitor cell lineage. We first compared the growth pattern of these cells treated with various concentrations of VPA against those without VPA treatment. At low concentrations (up to 500μM), VPA had no effect on the growth pattern of K562 cells. We then evaluated the effect of VPA on the efficiency of CRISPR guide RNAs to induce a double stranded break. A set of guide RNAs for two separate loci were evaluated. K562 cells treated with 50μM VPA 24 hours prior to nucleofection delivery of the guide RNAs were compared to untreated K562 cells. TIDE analysis, utilized to quantify cutting efficiency by identifying the indels that occur through the mutagenic DNA repair pathway of nonhomologous end joining, revealed an increase in cutting efficiency in the VPA treated cells. K562 cells were then treated with VPA concentrations ranging from 300mM to 0.5μM. Untreated K562 cells and VPA treated cells were then targeted with a donor DNA template containing a GFP reporter gene. GFP expression was evaluated using UV spectrophotometry and flow cytometry. Significant increases in percent GFP expression were observed in cells treated with 5μM, 50μM, 500μM, and 5mM VPA. A notable 25% increase in GFP expression was observed with the 500μM VPA treatment, 120 hours after nucleofection. Treatment with VPA can be expanded to virtually any experiment utilizing the CRISPR-Cas9 system to enhance gene targeting efficiency, and in the future, be used as a pretreatment for therapeutically relevant gene editing. We plan on repeating this experiment with Jurkat lymphoid cells, and eventually, hematopoietic stem and progenitor cells.
Poster #: 243
Campus: CSU Dominguez Hills
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Escherichia coli, Evolution, RNA-seq
Project Title: Characterization of the SspA Regulon of Escherichia coli in Long-Term Stationary Phase
Author List:
Gonzalez Carreon , Lizett; Graduate, Biology, California State University, Dominguez Hills, Presenting Author
Kram, Karin; Faculty, Biology, California State University, Dominguez Hills
Abstract: In Long-Term Stationary Phase (LTSP) of the Escherichia coli life cycle, cells experience stress caused by low nutrient availability, high levels of waste, and a high-pH environment. Advantageous mutations can appear in cells, which are then selected for because the cells are able to survive these conditions. One gene frequently mutated in long-term cultures is the sspA gene. Stringent Starvation Protein A (encoded by sspA), is expressed in order to help cells adapt to stressful environments by affecting expression of other stress-response genes. We have begun to characterize the SspA regulon, and how that regulon is affected by adaptive mutations in sspA. We used quantitative Polymerase Chain Reaction (qPCR) to monitor three genes previously reported to be regulated by SspA in two genetic backgrounds: wild-type (no mutation) and point mutant (containing a single nucleotide mutation originally identified as advantageous in LTSP). We monitored sspA expression along with the expression of cynX, hdeA, and fliC (flagellin), each potentially regulated by SspA. Comparing the point strain to the wild type strain, hdeA and fliC genes are upregulated in the point mutant strain, whereas cynX is downregulated in the point mutant strain. We also designed primers for qPCR analysis based on RNA-seq data which compared the wild-type and point mutant strain during 4 (log phase), 8 (late-log phase), and 24 (stationary phase) hours of growth, which identified potential novel targets of SspA. In the wild-type cells gadC, puuR, and ygiH are downregulated from 4 hours to 8 hours of growth while tdcA is up regulated. However, in the point mutant strain, genes are similar in this time period. Further, in wild-type cells gadC and puuR are upregulated from 8 hours to 24 hours of growth, and yqiH and tcdA are downregulated in contrast to the point mutant strain where puuR is upregulated while the rest of the gens are down regulated. These data indicate that the advantageous point mutation in SspA may be affecting genes early in the life cycle of E. coli, which leads to consequences in LTSP. Each of these genes, along with additional targets, will be monitored throughout growth into LTSP in order to further investigate the influence of SspA in the bacterial life cycle and adaptation to long-term cultures. Characterizing the role of SspA, along with the genes it regulates, during LTSP will lead to an understanding of how mutations influence adaptation to changing environments.
Poster #: 244
Campus: CSU Long Beach
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila, metabolism, development
Project Title: The Effects a High Fat Diet has on Drosophila melanogaster’s Fecundity and Metabolic Gene Expression
Author List:
Arciga, Ilse; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author, Nagel Award Nominee
Reyes, Carolina ; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Makary, Youstina; Graduate, Biological Sciences, California State University, Long Beach
Gago, Ingrid; Undergraduate, Biological Sciences, California State University, Long Beach
Eldon, Elizabeth ; Faculty, Biological Sciences, California State University, Long Beach
Abstract: Approximately two-thirds of the adult population in the United States is considered to be overweight or obese, meaning that around two-thirds of adults are likely to suffer from obesity-related illnesses such as diabetes, atherosclerosis, heart disease, high blood pressure and sleep disorders. Many of these diseases are common and preventable causes of death in the United States.
Drosophila melanogaster are used in this study as a model organism to examine the effects a high fat diet has at physiological and molecular levels over multiple generations. Drosophila melanogaster is an ideal model organism to study the effects of obesity due to its ease of breeding and maintenance, easily trackable developmental stages, and short lifespan. The short lifespan makes it ideal for multigenerational studies, allowing us to monitor the developmental effects of a high-fat diet on many generations of offspring.
We hypothesize that Drosophila melanogaster raised on a high fat diet will become obese, which will cause upregulation of genes involved in lipid metabolism and will also cause a decrease in fecundity, relative to flies raised on control food. We also hypothesize that differences in developmental timing will be observed. Vegetable shortening was added to the standard diet to increase its lipid content. Physiologically, the fecundity and lipid storage of D. melanogaster third instar larvae were observed. Molecularly, expression of the intestinal lipase gene, magro (mag), was studied. Initial results of triglyceride assays indicate a significant difference in the amount of lipid stored over multiple generations. Fecundity measurements for the 10th and 16th generations showed a significant difference between the different treatments. There were also significant differences in fecundity between the 10th and 16th generations. To detect molecular changes due to dietary fat, transcript levels for the intestinal lipase magro were measured by qPCR. Although larvae raised on vegetable shortening showed higher expression than those raised on a control diet, the difference was not statistically significant. We are currently investigating how different sources of fat affect lifespan and the timing of larval development to eclosion, which is known to be sensitive to nutrient levels. Our results indicate that a high fat diet affects the fecundity of Drosophila melanogaster over multiple generations.
We acknowledge CSUPERB and support by NIH RISE program grant R245GM071638.
Poster #: 245
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila, courtship, G-proteins
Project Title: Neurobehavioral Analysis of the Trapped in Endoderm 1 GPCR Pathway
Author List:
Tran, Cac; Graduate, Biological Sciences, San José State University, Presenting Author
Mu, Emily; Undergraduate, Biological Sciences, San José State University, Presenting Author
French, Rachael; Faculty, Biological Sciences, San José State University
Abstract: Understanding how genes direct neurodevelopment, which in turn specifies particular behaviors, is one of the main goals of neuroscience. Drosophila melanogaster is an excellent model for courtship behavior research. Male Drosophila perform an elaborate courtship ritual that consists of a series of stereotyped behaviors that must be performed correctly and in the right order in order to ensure female receptivity. These behaviors, while complex, are innate – male flies are born knowing how to perform them – but can be modified through environmental interactions (for example, rejection by the female). In addition, all of the steps of the ritual are established and regulated by the behavioral sex determination gene fruitless (fru). Thus, this set of behaviors provides an ideal model for the study of genetic and neural programming of behaviors.
We previously identified a novel function for the gene Trapped in endoderm 1 (Tre1) in courtship. Tre1 encodes for an orphan G-protein coupled receptor (GPCR) with roles in cell migration, as well as cell polarity determination in the developing nervous system. Tre1 is regulated by fru and required for normal courtship behavior. Neuronal loss-of-function of Tre1 results in an unusual courtship phenotype: male flies mutant for Tre1 initiate courtship on average twice as fast as wild type males. In addition, a subset of Tre1 mutant males fail to initiate courtship, indicating a complex requirement for this gene in courtship behavior. Given its role in germ cell migration and establishment of neuronal polarity, we hypothesize that Tre1 receives is involved in sex-specific neuronal pathfinding, and that disruption of this pathfinding leads to improper neuronal connectivity, culminating in altered courtship behavior.
We have begun characterizing the signal transduction pathway downstream of the Tre1 GPCR. In germ cell migration, Tre1 directs polarized downregulation of E-cadherin (encoded by shotgun, shg), resulting in its redistribution. We find that loss-of-function of shg results in significantly slower courtship initiation than that of wild-type, consistent with negative regulation of shotgun by Tre1
In addition, we have been characterizing the effects on courtship of mutations in genes encoding Drosophila G-proteins. We will present the results of testing four alpha subunits, one beta subunit, and two gamma subunits.
Poster #: 246
Campus: CSU Sacramento
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: marine invertebrate, molecular ecology, RNA extraction
Project Title: Developing Tissue Sampling and RNA Extraction Methods to Study Genetic Mechanisms of Thermal Tolerance in the Marine Snail Chlorostoma funebralis
Author List:
Ayala-Valdez, Lizvette; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Bedolla, Amanda; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Gleason, Lani; Faculty, Biological Sciences, California State University, Sacramento
Abstract: In this era of climate change, an understanding of the molecular mechanisms underlying individual thermal tolerance is needed. Previous work has shown that southern California populations of the marine snail Chlorostoma funebralis are more thermally tolerant and are exposed to high temperatures more often than northern California populations. In addition, the probability of reaching these stressful temperatures in the field is correlated with high control level expression of 222 genes in southern populations. However, to date gene expression and survival data have not been obtained from the same individuals; such a dataset would provide valuable insight into which of the 222 candidate genes actually confer a survival advantage under heat stress. The goal of this study was to develop nonlethal tissue sampling and RNA extraction protocols that can be used to identify correlations between gene expression and heat stress survival in individual C. funebralis. We tagged and measured the shell length of 22 C. funebralis and collected foot tissue samples from 14 adults; 8 individuals were used as handling or no-handling controls. We then monitored survival and growth of all individuals for 4 months to assess potential negative effects of tissue sampling. For RNA extraction, we homogenized each tissue sample using liquid nitrogen or mechanical disruption techniques and compared the quantity and quality of the resulting RNA. Tissue sampling did not cause mortality of any individuals, and an ANOVA test showed no significant growth differences between sampled and control individuals (p = 0.19). T-tests indicated the tissue samples homogenized by mechanical disruption resulted in higher RNA quantity (p = 0.01) and quality (p = 0.01) than those homogenized with liquid nitrogen. We thus conclude that these nonlethal tissue sampling, mechanical homogenization, and RNA extraction methods we have successfully developed are suitable for use in future studies to determine if expression of candidate genes is higher in C. funebralis individuals that survive vs. die after heat stress. Ultimately, enhanced genetic understanding of heat tolerance in marine mollusks can be used to identify and preferentially breed warm-water tolerant shellfish in aquaculture facilities and to reduce economic loss from global warming. CSU Sacramento, the CSU Council on Ocean Affairs, Science & Technology (COAST), and CSU Sacramento CIMERA ASI Funds provided financial support for this project.
Poster #: 247
Campus: Humboldt State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Genome editing, Molecular Genetics, Plasmids
Project Title: Development and Stable Integration of Tetracycline Inducible Tools for Genome Editing and Genome Regulation in Cultured Human Embryonic Kidney Cells
Author List:
Rodriguez, Miranda; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
Pope, Amanda; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
Mueller, Jasmine; Undergraduate, Biological Sciences, Humboldt State University
Villa, Miguel; Undergraduate, Biological Sciences, Humboldt State University
Steele, John; Faculty, Biological Sciences, Humboldt State University
Abstract: Our research lab uses CRISPR-based genome editing and gene regulator tools for functional genetic screens in cellular models of human neurodegenerative diseases. However, many of the cell types used by our lab have poor (<10%) transient transfection efficiency and readily suppress expression of viral promoters (e.g. CMV) that are typically used in transient expression plasmids. Therefore, we cannot reliably use many of the existing plasmids for transient transfection of these tools of our on-going genetic screens. The goal of this project was to develop a novel set of plasmid that: (i) can be used for targeted integration at the AAVS1 locus in the human genome and confer puromycin resistance, (ii) contain the TRE3G tetracycline-inducible promoter driving expression of the EGFP fluorescent protein with a cleavable linker, and (iii) a facile cloning site to insert CRISPR-based genome editing or regulatory tools.
To accomplish this goal, we constructed an AAVS1-repair plasmid containing a TRE3G tetracycline-inducible promoter that drives expression of the EGFP fluorescent protein with a T2A cleavable linker and cloning site for insertion of a second coding sequence that, once transcribed breaks the sequences there is two proteins--instead of having one long fused protein. When co-transfected with dual nickase CRISPRs targeting the AAVS1 locus, these tools can be stably integrated into the genome of human cells. Here, we produced a series of AAVS1-repair plasmids containing coding sequences for Cas9 D10A (nickase), dCas9 (enzymatically dead Cas9), dCas9-VP64 (transcriptional activator), or dCas9-KRAB (transcriptional repressor). These were cloned, assembled, screened by PCR, and our resulting plasmids were co-transfected into HEK293t human embryonic kidney cells along with our pLizzard plasmid (which contains the rtTA reverse tetracycline transactivator) and treated with doxycycline, successfully inducing expression of EGFP and the integrate CRISPR tools. We then used AAVS1-targeted dual nickase CRISPR gene editing to stably integrate these cassettes into the human AAVS1 locus on Chromosome 19. We showed that the resulting cell lines were stably puromycin resistant and able to induce expression of EGFP in the presence of doxycycline and the rtTA protein. These new tools will allow us to study more in depth on Huntington's disease, Alzheimer's disease , and genetic mutations of BRCA 1/2. Our next step is to integrate these into human neural progenitor cells.
Poster #: 248
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Sympathetic nervous system, Sensory Physiology, Muscle Spindle Afferents
Project Title: Sympathetic neurotransmitters decrease muscle spindle afferent sensitivity to muscle stretch in adult mice.
Author List:
Harnisch, Arthur; Graduate, Biological Sciences, San José State University, Presenting Author
Sanchez, Phylicia; Undergraduate, Biological Sciences, San José State University, Presenting Author
Wilkinson, Katherine; Faculty, Biological Sciences, San José State University
Abstract: Proprioception is the sense of body position in space, which is necessary for completing complex motor tasks. The most significant proprioceptors are the sensory neurons that innervate the muscle spindle, the Group Ia and Group II muscle spindle afferents (MSAs). The muscle spindle is a complex sensory organ located in parallel with contractile extrafusal muscle fibers. MSAs report changes in muscle length and muscle movement. The muscle spindle capsule is innervated by sympathetic neurons and adrenergic α1 receptors are found on intrafusal fibers. Overall, the importance and function of sympathetic innervation to the muscle spindle is not well understood.
We tested the hypothesis that sympathetic neurotransmitters (epinephrine, or EPI & norepinephrine, or NE) decrease the responsiveness of MSAs to stretch. We used an in-vitro mouse muscle-nerve preparation to record MSA firing activity. Briefly, the extensor digitorum longus (EDL) muscle and innervating deep peroneal branch of the sciatic nerve were dissected and perfused in an oxygenated synthetic interstitial fluid tissue bath. An extracellular recording electrode was placed on the nerve and individual MSAs identified by waveform. A series of 4s ramp-and-hold stretches were applied to the EDL and instantaneous firing frequency before and during stretch was measured. Upon exposure to NE (30 µM), there was a decrease in the firing rate at the beginning of the stretch (n = 3, 9% decrease) and at the end of the stretch (3.5s into stretch; n=3, 5 % decrease). To confirm dose dependence, 100uM NE showed a larger decrease at both the beginning (n= 2, 16% decrease) and end of the stretch (n=2, 25% decrease).
Upon exposure to EPI (30µM, or 100µM), there was also a decrease in response to stretch at the beginning (n = 3, 15% decrease) and end of stretch (n=3; 33% decrease). Larger decreases in firing were observed during the initiation of stretch with 100µM EPI, but was not observed during the maintenance of that stretch (0.5s into stretch; n=2; 31% decrease) (3.5s into stretch; n=2; 19% decrease). These results support the hypothesis that sympathetic neurotransmitters can act locally in the absence of central nervous system circuits to decrease the responsiveness of MSAs to stretch. Future studies will determine whether conditions that cause increased sympathetic nervous system activity can decrease MSA stretch sensitivity and compromise motor control.
Poster #: 249
Campus: Stanislaus State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: CRISPR, Mutagenesis, Plasmids
Project Title: Site Specific Mutagenesis in E. coli Plasmids using CRISPR
Author List:
Mocko, Cody; Undergraduate, Biology, California State University, Stanislaus, Presenting Author
Green, James; Undergraduate, Biology, California State University, Stanislaus, Presenting Author
Youngblom, Jim; Faculty, Biology, California State University, Stanislaus
Abstract: The purpose of this study is to determine whether an E. coli site-specific mutagenesis system using CRISPR in conjunction with recombineering (recombination-mediated genetic engineering) can be applied to mutate E. coli plasmids. We (and many others) have shown that CRISPR/recombineering can reliably mutate the E. coli chromosome; but CRISPR effects on plasmids have not been documented. To initiate the project, we constructed the plasmid pCRISPR::pCAS (containing the kanamycin resistant gene, KanR) that produces a guide RNA directed toward another plasmid- pCas9 (containing chloramphenicol resistance, CmR) that produces the Cas9 nuclease. Through electroporation of pCRISPR::pCAS into an E. coli recombineering strain harboring pCAS9 we were able to demonstrate that the Cas9 nuclease is cutting and eliminating the pCAS9 plasmid (i.e. when selecting on bacterial growth media the number of CmR colonies plummets). When the electroporation is repeated with the addition of a single-stranded 59-nucleotide DNA fragment with similarity that overlaps the nuclease target region in plasmid pCas9, severed plasmids are repaired by recombineering (i.e. the number of CmR colonies significantly increases). Integration of the 59 nucleotide DNA fragment saves the plasmid and can simultaneously introduce mutations- integration of the DNA fragment does not require 100% similarity to the plasmid. Our initial experiments introduced a 1 nucleotide site-specific mutation into the pCAS9 plasmid (confirmed by DNA sequencing). Current experiments are utilizing custom-made 59 nucleotide DNA fragments that are progressively more divergent- we are replacing larger plasmid segments (up to 11 nucleotides). Our long-term goal is to use this method for plasmid targeted gene replacement. This method could simplify construction of recombinant plasmids as the traditional steps involving restriction enzymes and DNA ligase are not required. Addition of small DNA fragments to your preferred plasmid could be as simple as ordering an oligonucleotide and performing an electroporation.
Poster #: 250
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: supercoiling, transcription, Caulobacter
Project Title: Gyrase-mediated Supercoiling Modulates the Expression of the Caulobacter crescentus sciP Promoter
Author List:
Requena, Kiana; Undergraduate, Biology, California State University, Northridge, Presenting Author
Heasley, Keaton; Alumnus, California State University, Northridge
Kumar, Priya; Alumnus, California State University, Northridge
Murray, Sean; Faculty, Biology, California State University, Northridge
Abstract: The location of genes on the Caulobacter crescentus chromosome is thought to regulate gene expression based on the methylation states of the promoters. As the replisome copies the chromosome, it leads to major changes in the methylation and supercoiling of DNA. We propose that DNA supercoiling, independent of promoter methylation state, controls the transcription of some cell-cycle regulated genes. We treated C. crescentus cultures with the antibiotic Coumermycin A1, a DNA gyrase inhibitor. As reported for other bacteria, high levels of the antibiotic inhibited growth, whereas low levels improved growth. SDS-PAGE analysis of cell lysates suggested a notable change in the presence of a few C. crescentus proteins under Coumermycin treatment, one of which is the master regulator SciP. There appeared to be no significant change in accumulation of the other cell cycle master regulators, suggesting that DNA supercoiling only regulates a subset of genes. Miller Assays and immunoblots confirmed that a decrease in sciP transcription leads to less SciP accumulation when gyrase activity is inhibited. We observed similar results in cells treated with the antibiotic and gyrase-inhibitor novobiocin. We identified plasmid that pJC326C can serve as a reporter for the degree of supercoiling in C. crescentus cells. When DNA supercoiling is inhibited, the plasmid relaxes and migrates more slowly through an agarose gel. We conclude that DNA supercoiling is important in the transcription of at least one cell cycle master regulator in C. crescentus.
This work was supported by NIH R25 GM063787 to MariaElena Zavala in support of KH, NIH 8TL4GM118977-02 to Crist Khachikian in support of PK and KR, a CSUN RSCA Award to SM, and NIH grant SC3 GM121234 to SM.
Poster #: 251
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: gene regulatory network, neural crest, evolution
Project Title: Dissecting the relationship between Sox10 and the neural crest gene regulatory network in two vertebrate species
Author List:
Camacho-Avila, Alexis; Undergraduate, Biology, California State University, Northridge, Presenting Author
Rogers, Crystal; Faculty, Biology, California State University, Northridge
Abstract: Neural crest (NC) cells are embryonic stem-like cells that give rise to many derivatives including craniofacial bone and cartilage, pigment cells and most of the peripheral nervous system. These cells arise in the dorsal neural tube, but then undergo an epithelial to mesenchymal transition (EMT) to migrate to numerous destinations. Understanding the molecular mechanisms that regulate how these cells are created and the proteins that control their development is crucial to identify possible causes for congenital defects such as cleft palate, albinism, and Hirschsprung’s Disease. Several transcription factors have been identified as important mediators of NC specification and EMT. Here, we perform comparative analysis of NC development in two divergent vertebrate species, the salamander, Ambystoma mexicanum (axolotl), and the amniote, Gallus gallus (chicken). We characterized multiple NC transcription factors and identified that while the spatiotemporal expression of the factors is conserved, the NC gene regulatory network (GRN) may not be. Pax7 is expressed in the dorsal neural tube and pre- and post-migratory NC cells, and Sox9 is only expressed in a subset of Pax7-positive cells in both organisms. Gain and loss of function studies were used to determine if the function of the NC transcription factors is conserved. We identified that in chick embryos, overexpression of Sox10 is sufficient to induce ectopic expression of Sox9, but not Pax7. However, in axolotls, chicken Sox10 can activate Pax7 as well as Sox9 suggesting possible species-specific developmental differences. These studies will increase our understanding of the similarities and differences that vertebrate species use to regulate NC development, and will provide new insight into potential therapeutic approaches by highlighting new tools that can be used across the vertebrate spectrum.
Poster #: 252
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila, aging, ethanol
Project Title: Developmental Ethanol Exposure Slows Down the Aging Process in Drosophila Melanogaster
Author List:
Belhorma, Khaoula; Graduate, Biological Sciences, San José State University, Presenting Author
Darwish, Nahed; Graduate, Biological Sciences, San José State University, Presenting Author
French, Rachael; Faculty, Biological Sciences, San José State University
Abstract: Alcohol is a known teratogen that results in a spectrum of disorders known as Fetal Alcohol Spectrum Disorder (FASD). Children born with FASD can exhibit a range of symptoms such as low birth weight, microcephaly and neurobehavioral problems. FASD costs the United States alone 4 billion dollars every year and is estimated to cost 2 million dollars per individual’s lifetime.
We have established Drosophila melanogaster as a model organism for the study of FASD. We found that several alleles of dementin (dmtn), the Drosophila ortholog of the Alzheimer Disease associated protein TMCC2, convey sensitivity to develomental ethanol. In addition, quantitative reverse-transcriptase mediated PCR (RT-qPCR) analysis demonstrates that dmtn transcription is decreased an average of 64% in larvae reared in ethanol, indicating that dmtn expression is disrupted by ethanol.
In fies, the ability to climb is correlated with central nervous system health. In addition, the gradual loss of climbing ability is a well-established fly model for aging. We therefore tested nervous system function in ethanol-reared flies via negative geotaxis (climbing) assays.
We find that wild type flies reared on ethanol exhibit mild climbing defects suggestive of neurodegeneration. Surprisingly, we also find that flies reared in ethanol age more slowly than control animals, as do dmnt mutant flies.
We hypothesize that the reduced aging seen in ethanol-reared flies may be due to the reduction in dmnt expression in these animals. We will test this hypothesis by attempting to rescue the slow aging of ethanol-reared flies through transgenic overexpression of dmnt. In addition, we are currently testing known aging mutants for their sensitivity to developmental ethanol exposure. Finally, we have previously shown that ethanol exposure during development causes increased expression of antioxidant genes, and we are testing the hypothesis that persistent upregulation of these genes in adult animals contributes to their slow aging.
In conclusion we have found that, in addition to the well-documented negative effects of developmental alcohol exposure on the nervous system, there may be a previously-unsuspected neuroprotective effect of developmental ethanol exposure. We will present the results of experiments to test the hypothesis that this protective effect is mediated through a combination of downregulation of the dmtn gene and induction of antioxidant genes.
Poster #: 253
Campus: San Diego State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Dengue virus, cell-based assays, protease inhibitor screens
Project Title: Coupling of two cell-based counter screens to reveal potential antivirals against Dengue virus
Author List:
Slemons, Danielle; Graduate, Biology, San Diego State University, Presenting Author
Canter, Benjamin; Undergraduate, Biology, San Diego State University, Presenting Author
Smurthwaite, Cameron; Staff, Biology, San Diego State University
O’Hanlon, Ryan; Graduate, Biology, San Diego State University
Wolkowicz, Roland; Faculty, Biology, San Diego State University
Abstract: Many viruses require proteolytic cleavage, either by viral or host proteases, for maturation and full activity of their proteins. Viral proteases have thus been obvious targets for the development of antivirals. Our lab previously developed a cell-based assay that monitors viral protease activity focusing on HIV-1 protease (PR) as proof of principle. The assay is based on the inducible expression of the PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the green fluorescent protein (GFP) downstream reporter only in the presence of an effective PR inhibitor. For Dengue virus (DenV), the causative agent of DenV Shock Syndrome and Hemorrhagic Fever, there is no treatment other than bedside care as no antivirals are currently available on the market. We have thus adapted the HIV-1 PR assay to the DenV PR non-structural protein 3 (NS3). DenV NS3 is a serine PR that requires a co-factor (NS2B) for enhanced proteolytic activity and for stabilizing proper folding. NS2B has four transmembrane domains (TM), two on either side of a hydrophilic cytoplasmic-exposed region. In order to monitor the activity of DenV PR in the context of the assay (referred to as cytosolic assay), we needed to remove the NS2B TMs to allow the fusion protein to travel to the nucleus. We have thus engineered a wild type and mutant NS2B/NS3, and compared full-length with delta-TM counterparts. Flow cytometry and western blotting corroborated activity and thus lack of fluorescence only with the wild type PRs. As this assay is ultimately intended for drug discovery, we have also developed a secondary novel assay that can be used as a counter screen. This assay utilizes Gal4 and GFP in a novel manner, where rather than deleting the TMs of NS2B, we exploit them to anchor our constructs in the Endoplasmic Reticulum membrane. For this assay, referred to as anchored assay, we engineered an intact Gal4 downstream NS2B/NS3 and a well-known NS3 cleavage site. Gal4 is released upon cleavage, thus activating GFP only when PR is active. We have recently obtained cell lines for the cytosolic assay and we are in the process of obtaining similar clones for the anchored assay. The combination of assays with opposite readouts (in the cytosolic assay green fluorescence appears only with the mutant PR while in the cytosolic assay only with wild type) will increase the potential of finding inhibitors in future drug screens.
Poster #: 254
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: XoxF, PQQ, lanthanide-switch
Project Title: Pyrroloquinoline quinone is required for lanthanide-dependent methanol dehydrogenase expression in Methylobacterium extorquens
Author List:
Hoeber, Caitlin; Undergraduate, Biological Sciences, San José State University, Presenting Author
Nguyen, Mona; Undergraduate, Biological Sciences, San José State University, Presenting Author
Valentine Crisostomo, Ralph; Graduate, Biological Sciences, San José State University
Kaur, Simi; Graduate, Biological Sciences, San José State University
Skovran, Elizabeth; Faculty, Biological Sciences, San José State University
Abstract: Methylobacterium extorquens is a model organism for the study of methylotrophic growth and is of interest for the production of value-added chemicals from methanol and formate, which are inexpensive feedstocks that can be electrocatalytically generated from waste CO2 and renewable electricity. M. extorquens can oxidize methanol via a calcium (Ca)-dependent methanol dehydrogenase (MxaFI) or a lanthanide (Ln)-dependent methanol dehydrogenase (XoxF). The addition of exogenous Ln results in repression of mxaFI and upregulation of xoxF, a regulatory process that has been named the Ln-switch. The regulatory network that controls the Ln-switch is complex and involves at least two sensor kinases, three response regulators, and a LysR-type transcriptional regulator. It is not yet known what signals other than Ln contribute to the Ln-switch or how this complex regulatory network operates. To identify additional proteins and cellular processes required for expression of xoxF, transposon mutagenesis was used in combination with a fluorescent xoxF transcriptional reporter fusion. Four clusters of genes were identified as required for expression of xoxF including two operons required for pyrroloquinoline quinone (PQQ) biosynthesis, a putative Ln transport gene cluster, and a putative cytosol aminopeptidase, META1_3908. Mutations in each gene cluster were reconstructed and expression from the xoxF promoter was measured. Loss of genes from each identified cluster resulted in repression from the xoxF promoter. To verify that PQQ was a signal for xoxF expression, exogenous PQQ was added to the growth medium, which rescued expression of xoxF in the PQQ biosynthesis mutants. Interestingly, PQQ also rescued expression in the META1_3908 mutant. Growth analysis showed that the growth defects observed for the META1 3908 mutant could be partially rescued by exogenous PQQ suggesting that META1_3908 contributes to PQQ synthesis or utilization. We also show that like xoxF, expression from the pqqA promoter is affected by Ln, PQQ, and the MxbDM two-component system. This work identifies PQQ as a new signal that contributes to the regulation of the Ln-switch, identifies a novel gene that contributes to PQQ biosynthesis or use, demonstrates that PQQ biosynthesis is also under control of the Ln-switch, and adds knowledge about the regulation of a crucial step in methylotrophic metabolism. Funding for this work was provided by San Jose State University and CSUPERB.
Poster #: 255
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: lysosomes, ion channel, brain diseases
Project Title: Targeted induction of MCOLN2 and MCOLN3 Gene Expression using Drug Compounds
Author List:
Sanchez, Vanessa; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Cuajungco, Math; Faculty, Biological Science, California State University, Fullerton
Abstract: Mucolipidosis type IV (MLIV) is a devastating neurodegenerative disease caused by mutations or deletions within the Mucolipin-1 (MCOLN1) gene, which encodes the TRPML1 protein. The clinical manifestation of MLIV includes cognitive and motor deficits, cataract formation, and blindness. At the cellular level, the recycling compartments called lysosomes accumulate lipids and substances that potentially trigger cell death. TRPML1 is a non-selective cation channel related to TRPML2 and TRPML3, encoded by MCOLN2 and MCOLN3, respectively. These three proteins play a key role in membrane trafficking and endosome-lysosome fusion. In addition, they are known to interact with each other and form a functional heteromeric ion channel. TRPML1 and TRPML2 share a high degree of sequence similarity and ion channel characteristics. Currently, there is no cure or treatment for MLIV, and most MLIV patients do not have a functional TRPML1. Since TRPML2 closely resembles TRPML1, we hypothesize that TRPML2 may be able to substitute for the loss of TRPML1 function in MLIV by increasing TRPML2 expression. A previous drug screening of over 800 drug compounds from the National Institutes of Health (NIH) Clinical Collection revealed candidate drugs that upregulate endogenous MCOLN2 or MCOLN3 transcripts in cultured human cell lines. To further validate the effects of drug candidates, we cultured human HeLa and HEK-293 cells and exposed them at varying drug concentrations to determine the minimal effective concentration (MEC). Using standard polymerase chain reaction (PCR) and real-time quantitative PCR, we found that endogenous MCOLN2 and MCOLN3 transcripts were up-regulated by the first drug candidate at 100 micromolar, the second at 300 micromolar, and the third at 2 micromolar concentration. Although we did not observe cytotoxicity at these concentrations, additional experiments are ongoing to determine whether the MEC affects cell viability. Identification of an efficacious drug candidate to induce TRPML2 expression and rescue the loss of TRPML1 can guide us towards a pre-clinical evaluation using a Mcoln1 knockout mouse model. This study will provide insight into whether TRPML2 or TRPML3 could be used as a therapeutic substitute for patients suffering from MLIV disease.
Poster #: 256
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: DNA replication, translesion synthesis, DNA damage
Project Title: Regulation of translesion synthesis polymerases eta and kappa in response to DNA replication stress
Author List:
Eruaga, Isaiah; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Huang, Tony; NYU Langone Health, NYU Sackler Institute of Graduate Biomedical Sciences, Associate Professor
Abstract: The cell faces constant pressures from exogenous and endogenous factors that damage DNA. During DNA replication, when the replisome encounters damaged DNA it causes replication stress – the slowing or stopping of DNA replication machinery. Excessive replication stress can cause a host of complications like gene translocations and apoptosis. Thus, the cell has developed a mechanism to tolerate DNA damage during DNA replication: translesion synthesis (TLS) polymerases that are able to bypass DNA lesions, allowing the replisome to replicate damaged DNA. The Y-family polymerases are a group of TLS polymerases which are functionally and structurally similar but exhibit preferences for different types of lesions. For instance, lesions caused by UV light exposure are bypassed by TLS polymerase Eta, and our group’s recent work has shown that replication stress caused by nucleotide depletion is handled by TLS polymerase (Pol) Kappa. However, the mechanisms that regulate TLS polymerase usage are not well understood. To begin to elucidate how the usage of Y-family TLS polymerases Eta and Kappa are regulated in response to replication stress, this study exposed mammalian cell lines HEK 293T and RPE to replicative stressors, UV light or hydroxyurea (a drug which reduces nucleotide levels). Then western blotting was performed to analyze the effect of these treatments on TLS polymerase expression and various markers for cell stress and DNA damage. In response to UV, the expression of the TLS polymerase responsive to nucleotide depletion, Pol Kappa was reduced. We hypothesize that this may preclude Pol Kappa from competing with the more efficient and accurate UV lesion bypass by Pol Eta. The use of a proteasome inhibitor, MG132, indicated that loss of Pol Kappa was due to proteasome-dependent degradation. Moreover this effect was seen in both mammalian cell lines tested. Pol Eta was also shown to undergo a proteasome-dependent degradation in RPE cells. To our knowledge, this study is the first to show that Pol Kappa-specific degradation may regulate TLS polymerase usage. Translesion synthesis is an integral part of the DNA damage tolerance pathway, and understanding the regulation of this phenomenon is relevant to cancer and the design of treatments that could help potentiate existing genotoxic mainstay cancer therapies: chemotherapy and radiotherapy. Funding from NYU Sackler Institute of Graduate Biomedical Sciences Summer Undergraduate Research Program.
Poster #: 257
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: premetastatic niche, breast cancer, stromal cell-cell crosstalk
Project Title: Metastatic Breast Cancer Secretomes are Enriched for an LCN2 Axis and Induce Differential Stromal Remodeling within the Lung and Brain
Author List:
Aguayo, Analine; Undergraduate, Biology, California State University, Northridge, Presenting Author, Nagel Award Nominee
Meade, Kayla; Graduate, Biology, California State University, Northridge
Sanchez, Francesca; Graduate, Biology, California State University, Northridge, Presenting Author
Nadales, Nathalie; Undergraduate, Biology, California State University, Northridge
Hamalian, Sarkis; Graduate, Biology, California State University, Northridge
Uhlendorf, Toni; Staff, Biology, California State University, Northridge
Banner, Lisa; Faculty, Biology, California State University, Northridge
Kelber, Jonathan; Faculty, Biology, California State University, Northridge
Abstract: Metastatic spread of tumors accounts for over 95% of cancer-related deaths. Before cancer cells can survive in distant microenvironments, these tissues must be primed to acquire tumor-permissive properties. The primary tumor communicates with the premetastatic niche, or molecular environment which tumor cells may eventually metastasize to, by releasing factors that recruit cells, such as mesenchymal stem cells (MSCs) and macrophages (MACs), to be reprogrammed and support this tumor-permissive environment. However, the cell-cell communication that occurs during premetastatic niche priming is poorly understood. To address this, we employed in vivo and in vitro methods to evaluate how secretomes from high/low-grade metastatic/non-metastatic mouse and human breast cancer cells (BCCs) may promote MSC recruitment and macrophage polarization. We show that secretomes from metastatic BCCs remodel lung and brain tissues in a manner that may support cancer cell seeding and expansion, with evidence of MSC-macrophage cross-talk only being observed in the brain, through IHC/IF staining of tissues/cells of mice chronically intraperitoneal injected with media conditioned by high-/low-grade BCC secretomes. Furthermore, we show that secretomes from BCC-educated MSCs or macrophages/monocytes increase pro-tumorigenic, anti-inflammatory cell states of macrophages/monocytes or MSCs, respectively. Finally, analysis of secreted factors differentially produced by metastatic BCCs revealed an LCN2 axis that predicts poor survival in breast cancer patients. These results indicate that cell-cell crosstalk may mediate reprogramming of the premetastatic niche and justify developing targeted therapeutics to inhibit this cross-talk.
Supported By: CSUN CSM and ORSP, CSUPERB, NIH (5SC1GM121182-02 to J.A.K.) and Research Initiative for Scientific Enhancement (R25GM063787 to M.E.Z. and A.A.) and the Sidney Stern Memorial Trust.
Poster #: 258
Campus: CSU Long Beach
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila, cell migration, organogenesis
Project Title: Analyzing Interactions between the 18-Wheeler Gene and X-linked Genes in Drosophila Salivary Gland Development
Author List:
Villalba, Jaqueline; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Molina, Anel; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Eldon, Elizabeth; Faculty, Biological Sciences, California State University, Long Beach
Abstract: Drosophila melanogaster salivary glands are an excellent model for mammalian organ development. The 18-wheeler gene is expressed during embryonic development where it regulates epithelial cell signaling and migration. To identify genes that interact with 18-wheeler during development we take advantage of the observation that an embryo heterozygous for both 18-wheeler and a gene that interacts with18-wheeler will produce defective salivary glands. A collection of 93 X-chromosome-linked deficiencies (Df(1)) is used to search for interactions with 18-wheeler. Together these deficiencies delete 2,288 of the 2,331 euchromatic genes on the X-chromosome (98.1%). To obtain embryos that are heterozygous for 18-wheeler and an X-linked deficiency, males carrying an 18-wheeler mutation and a green fluorescent protein (GFP) reporter expressed in salivary glands (stock 84-1) are mated with females heterozygous for an X-linked deficiency. Their other X is a GFP-expressing balancer chromosome. Control embryos are obtained by using males that are wild type at the 18-wheeler locus, but still carry the GFP salivary gland reporter (stock 15-1). Embryos are collected, fixed, and used for immunocytochemistry to detect GFP, which is expressed in the salivary glands of the Df(1); 18-wheeler embryos. If the mutations interact, salivary gland morphogenesis will be abnormal. Defects include, but are not limited to, glands lengthening, shortening, or migrating asymmetrically. Df(1)BSC719 (stock 26571) shows a gene interaction, but not in the manner predicted. When crossed to the 18-wheeler mutant, wild type glands are observed, but when crossed to wild type 18-wheeler, the glands possess morphological differences in migration pattern and development. This suggests that the deficiency causes a defect in gland morphogenesis that is rescued by reducing the dosage of the 18-Wheeler protein. Df(1)ED7374 (stock 8954) shows a genetic interaction. Heterozygous embryos for the deficiency and mutant 18-wheeler express abnormalities in migration and morphology. The homozygous 18-wheeler wildtype and heterozygous deficiency have wildtype glands. We conclude that this deficiency contains gene(s) that may interact with 18-wheeler. Current work focuses on using smaller deficiencies that map within Df(1)BSC719 and Df(1)ED7374 to identify the gene(s) that interact with 18-wheeler during salivary gland morphogenesis.
This research was supported by NIH-GMS 5UL1GM118979; 5TL4GM118980; 5RL5GM118978.
Poster #: 259
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: leukemia, cell death, gene regulation
Project Title: The Role of E4BP4 in Apoptosis of Human Leukemia Cells in Culture
Author List:
Mitchell-Velasquez, Erick; Undergraduate, Biology, California State University, Northridge, Presenting Author
Gomez, Danny; Graduate, Biology, California State University, Northridge
Medh, Rheem; Faculty, Biology, California State University, Northridge
Abstract: Apoptosis is a physiological and genetically controlled process of cell death important for development and cellular homeostasis. Several diseases can be attributed to an imbalance between cellular proliferation and attrition, brought on by inadequate or excessive apoptosis. In cancer, a lack of apoptosis causes excessive accumulation of unwanted cells. Apoptosis, or type I programmed cell death, is primarily regulated via altered expression of key genes and modulation of signal transduction pathways. Research in our laboratory focuses on understanding the early apoptosis events triggered by chemotherapeutic agents in cell culture models of leukemia. Previous studies have shown that the E4BP4 (adenovirus E4 binding protein 4) gene in upregulated in correlation with apoptosis. E4BP4 upregulation has been implicated in induction of the pro-apoptotic BIM protein, a member of the Bcl-2 family of proteins. In studies presented here, we have utilized CRISPR-mediated genomic editing technology to knock down E4BP4 expression. Three clones with reduced E4BP4 expression were analyzed for their response to Dexamethasone (Dex), a synthetic glucocorticoid and anti-leukemic agent. While E4BP4 knockdown did not alter the ability of cells to undergo apoptosis in response to Dex, two of the three clones showed a reduction in the extent of BIM upregulation. Residual E4BP4 expression in our knock down clones may be sufficient to evoke an apoptotic response. Further efforts are underway to understand downstream pathways regulated by E4BP4.
Acknowledgements: This research is funded by a NIH SCORE SC3 award to RDM (GM081099) and a MARC-U*STAR scholarship to EM.
Poster #: 260
Campus: San Diego State University
Poster Category: Other
Keywords: Photocatalysis, Electroactive Bacteria, Renewable Energy
Project Title: The First Generation of the Self-sustaining Microbial Photoelectrosynthesis for Hydrogen Generation and Waste Water Cleaning
Author List:
Williams, Nicholas ; Graduate, Department of Chemistry & Biochemistry, San Diego State University, Presenting Author
Patrick, Maggie; Graduate, Department of Chemistry & Biochemistry, San Diego State University, Presenting Author
Gu, Jing; Faculty, Department of Chemistry & Biochemistry, San Diego State University
Abstract: Sunlight offers an inexhaustible and sustainable source of renewable energy to meet our increasing energy demand. However, direct harvesting of solar energy is still challenging due to the variation of natural sunlight and its intermittent nature. To meet the Terawatt Challenge and harness large quantities of energy, current artificial photosynthesis systems are showing great promise for their capability of imitating natural photosynthesis. However, direct water splitting by artificial photosynthesis requires enormous energy input. Here, we demonstrate GaInP2-TiO2-MoSx, a photocathode, could couple with a microbial electrochemical oxidation process, to combat this energy issue and treat wastewater. The electroactive bioanode, composed of a natural mixed culture deposited on carbon fiber brush, can recover the chemical energy embedded in organic molecules, via oxidation, to form electricity, carbon dioxide (or bicarbonate) and protons. The electricity and proton produced is then utilized by the photocathode to produce hydrogen gas, a clean energy carrier. The production of hydrogen gas was confirmed via as chromatography, and the photocathode-bioanode system was found to be stable for 24 hours, limited by the photocathode. This self-sustaining system could generate a current of 0.4 mA/cm2 under 1 sun-power without any external bias using artificial waste water. We found that by coupling electroactive bacteria and a stable light absorbing material that could efficiently produce hydrogen gas with the potential to simultaneously clean waste water of organics. These studies were done in collaboration with Dr. Lu Lu and Dr. Jason Ren of Princeton University, with the help of funding from the National Science Foundation award.
Poster #: 261
Campus: CSU Sacramento
Poster Category: Other
Keywords: Neurodevelopment, Drosophila, Autism
Project Title: The Environmental Neurotoxicant Polychlorinated Biphenyl-95 Phenocopies a Common Autism Risk Gene in Drosophila melanogaster
Author List:
Murphy, Lillian; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author, Nagel Award Finalist
Nguyen, Kimberly; Undergraduate, Biological Sciences, California State University, Sacramento
Stryder, Brandon; Undergraduate, Biological Sciences, California State University, Sacramento
Ghenta, Kristina; Undergraduate, Biological Sciences, California State University, Sacramento
Welch, Chloe; Undergraduate, Biological Sciences, California State University, Sacramento
Mulligan, Kimberly; Faculty, Biological Sciences, California State University, Sacramento
Abstract: BACKGROUND: The interaction of environmental chemicals with specific genetic susceptibilities is linked to autism spectrum disorder (ASD). However, little is known about specific environmental chemicals that interact with genes to cause ASD. There are over 80,000 industrial chemicals that can be found in our environment and most have undergone little to no toxicological data. Therefore, the field urgently needs an efficient method for chemical analysis. This project involves the development of assays using Drosophila melanogaster for identification of chemicals that molecularly converge with Drosophila fmr1 (fragile X mental retardation 1). Mutations in human FMR1 are the most common single gene cause of ASD and the role FMR1 plays in neural development is conserved from flies to vertebrates. Data from vertebrate model organisms suggest that gestational exposure to the environmental neurotoxicant, polychlorinated biphenyl 95 (PCB-95), can increase the risk of ASD. Loss of Drosophila fmr1 causes a decreased courtship index (CI; a quantitative measure of innate courtship behavior) and impaired axon outgrowth in the adult brain. METHODS AND RESULTS: We exposed developing fruit flies to PCB-95 and used the courtship assay to determine that exposure to nanomolar concentrations of PCB-95 significantly decreases the CI in wild-type (wt) flies in a dose-dependent manner, and exhibits a synergistic reduction in the CI of fmr1 flies. We used immunohistochemistry and confocal microscopy to examine adult brains and found a dose-dependent increase in axon-outgrowth defects in the mushroom body (MB) of wt flies, which is the same phenotype caused by loss of fmr1. This finding suggests that PCB-95 may confer increased risk of ASD in individuals with FMR1 mutations—they affect the similar neurodevelopmental processes indicating potential molecular convergence in developing neurons. FUTURE DIRECTIONS: We are currently examining axon outgrowth in fmr1 flies following PCB-95 exposure to determine if a synergistic response occurs with the axon outgrowth phenotype. FUNDING: California State University Program for Education and Research in Biology (CSUPERB) New Investigator Award.
Poster #: 262
Campus: San Diego State University
Poster Category: Other
Keywords: Atomic layer deposition , biomaterial development , functionally graded materials
Project Title: Gas phase infiltration of porous materials via Atomic layer Deposition – a great opportunity for functional biomaterial development
Author List:
Yang, Fan; Postdoc, Department of Chemistry, San Diego State University, Presenting Author
Fairchild , Michael ; Undergraduate, Department of Chemistry , Presenting Author
Gu, Jing; Faculty, Department of Chemistry & Biochemistry, San Diego State University
Abstract: Atomic Layer Deposition (ALD) is a gas phase thin film deposition technique with accuracy down to nanometer or even angstrom scales. In a typical ALD process, gas phase precursors alternatively interact and chemically bond to the solid substrate surface until its reach a self-saturation state. However, if the substrate is a porous or soft material, a gaseous precursor tends to penetrate into the deep inside of the substrate. Working in the gas phase enables the application of this synthetic method, which involves building functionally gradient interfaces in a porous material. This type of interface allows metal active sites to progressively vary in one or more dimensions. This interface will demonstrate a gradual change in the chemical, mechanical and catalytic properties as a function of position. Man-made functionally graded materials (FGMs) are commonly used in biomedical and optoelectronic devices such as man-made bones. With sufficient time and space for diffusion, the gaseous precursor will be able to penetrate into porous materials and complex 2D or 3D structures.
In our study, a Zinc based volatile precursor (diethyl Zinc) is used to interact with a Tetrakis(4-carboxyphenyl)porphyrin (TCPP) based metal-organic framework (MOF) in an ALD chamber, since porphyrin is one of the common but important units in a biological system. Strong evidence from X-ray diffraction, X-ray photoelectron spectroscopy (XPS) and scanning transmission electron microscopy (STEM) prove that Zinc precursor is successfully infiltrated into the porous MOFs, creating Zn-porphyrin metalation sites. The processed Zn-MOF sample showed superior electrochemical catalytic reactivity with 99% selectivity of reducing CO2 into CO, outperforming its counterpart that is performed with a traditional solution-based method and the unprocessed MOF sample. For a next step, X-ray absorption near edge structure (XANES) technique will be carried out to help in understanding the reason for the improved catalytic reactivity. Through this demonstration, we believe the ALD processing method has great potential for the modification of soft materials especially biomaterials for various bio-applications.
Poster #: 263
Campus: CSU Fresno
Poster Category: Other
Keywords: acoustic surveying, bat monitoring, ecosystem services
Project Title: Using biotechnology to choose bat box deployment sites: the role of structural and acoustic complexity
Author List:
Graves, Chrisionna; Undergraduate, Biology, California State University, Fresno, Presenting Author
Reece, Joshua; Faculty, Biology, California State University, Fresno, Presenting Author
Abstract: Bats are a biodiverse group of mostly insectivorous mammals that confer more than $22.9 billion per year of ecosystem services to our economy through the consumption agricultural pests. Providing man-made bat boxes provides roosting habitat and encourages bat presence near fields that benefit from the agricultural ecosystem services that bats provide. However, the success of bat boxes depends on a number of features and is currently as much an art as a science. Bats have certain well studied preferences for roosting such as box shape, height, and hours of direct sunlight. However, their preference for structural complexity in the surrounding environment is not well characterized; nor is there a systematic assessment of bat preference for noisy versus quiet environments, despite the fact that bats navigate and hunt largely acoustically. We used published studies of bat box deployment to systematically deploy 60 bat boxes in paired urban areas in Fresno: California State University Fresno campus and the Fresno Chaffee Zoo. Our goal was to improve bat box placement success by integrating traditional strategies with modern biotechnology. At each location, we measured the structural complexity of the environment using 360 degree digital images and novel software for pixelating and characterizing the structural complexity of the image. We made recordings of ambient noise at each location to assess common dimensions of the soundscape (sound frequencies, heterogeneity, intensity). We analyzed these data and depict correlations between structural and acoustic complexity and the influence of structure and acoustics on bat presence. We find that 1) structural complexity strongly positively influences acoustic complexity, and 2) that bats occur at higher frequencies when ambient sound intensity is low at the frequency at which they echolocate. Our data suggest that structural and/or acoustic complexity and characteristics are an important dimension to consider when strategically placing bat boxes and that ambient sound intensity from human may depress bat presence.
Poster #: 264
Campus: CSU Sacramento
Poster Category: Other
Keywords: Neurodevelopment, Drosophila, Autism
Project Title: Heterozygous loss of kismet, the Drosophila ortholog of an autism-risk gene, affects axon outgrowth and courtship phenotypes in adult fruit flies
Author List:
Welch, Chloe; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author, Nagel Award Finalist
Hu, Alain ; Undergraduate, Biological Sciences, California State University, Sacramento
Lew, Amy ; Graduate, Biological Sciences, California State University, Sacramento
Murphy, Lillian ; Undergraduate, Biological Sciences, California State University, Sacramento
Nguyen, Darren ; Undergraduate, Biological Sciences, California State University, Sacramento
Mulligan, Kimberly; Faculty, Biological Sciences, California State University, Sacramento
Abstract: BACKGROUND: Autism spectrum disorder (ASD) refers to a group of heterogeneous neurodevelopmental disorders that afflict 1 in 59 children in the United States. Genomic studies indicate that mutations in chromodomain helicase DNA binding protein 8 (CHD8) are one of the strongest risk factors for ASD. CHD8 is a chromatin modifier that influences the transcription of many other ASD-risk genes; thus, it is regarded as a master regulator that defines a common ASD subtype, characterized by macrocephaly and gastrointestinal (GI) problems. Yet, the cellular phenotypes caused by CHD8 mutations have not been completely explored. The overarching goal of this research project is to define both neural and GI phenotypes in Drosophila to elucidate how mutations in CHD8 impair development to cause ASD. The experimental approach described here will ultimately be used to study connections between gut microbiota and neural phenotypes in fruit flies. METHODS AND RESULTS: The Drosophila ortholog of CHD8, kismet (kis), is expressed throughout development and complete loss of kis is embryonic lethal, so we examined heterozygous kis mutants. We used immunohistochemistry and confocal microscopy to show that heterozygous loss of kis causes severe axon outgrowth defects in the mushroom body, an adult neural structure required for learning and memory. Disrupted axon outgrowth is one of the cellular phenotypes common to ASD. We also found functional phenotypes using the courtship assay, a quantitative measure of innate courting behaviors in Drosophila; heterozygous kis mutants exhibit significant reductions in courtship index, number of copulation attempts, and percent successful copulation. These results support kis playing a critical role during neural development, and they will also allow us to quantitatively assess how variations in gut microbiota affect the severity of kis phenotypes. FUTURE DIRECTIONS: We are currently examining relative phylogeny and abundance of metabolic gene clusters for gut microbiomes of wild-type versus kis Drosophila and will examine how changes to gut microbiota influence neural phenotypes. We are also investigating cellular phenotypes within the gut epithelium in an effort to understand how loss of kis may influence changes in the gut microbiome and cause GI problems associated with the CHD8 subtype of ASD. FUNDING: California State University Program for Education and Research in Biology (CSUPERB) New Investigator Award.
Poster #: 265
Campus: CSU Long Beach
Poster Category: Other
Keywords: wastewater, qPCR, activated sludge
Project Title: Monitoring Foaming Bacteria’s (Gordonia amarae) Growth in a Partially Nitrifying Water Reclamation Plant
Author List:
Asvapathanagul, Pitiporn; Faculty, Civil Engineering and Construction Engineering Management, California State University, Long Beach, Presenting Author
Nguyen, John; Undergraduate, Civil Engineering and Construction Engineering Management, California State University, Long Beach
Abstract: Usable water in Southern California’s dry climate is very scarce, so it must be recycled. Water Reclamation Plants (WRPs) convert wastewater into usable forms for irrigation, toilets, and etc. Nevertheless, frequently solids separation problems (foaming incidents) accidentally occur in activated sludge, which lowers quality of reclaimed water and increases energy, labors and chemical costs to eliminate foams. Overgrowth of filamentous bacteria causes foaming incidents. Our study goals are to identify the foaming bacteria and to discover the causes of the overgrowth of filamentous bacteria in the water samples. The wastewater samples were obtained from a partially nitrifying wastewater plant located in Southern California. Molecular techniques, including gene amplification (PCR), gene amplification (qPCR) and gene sequenceing, combined with statistical analysis prior to determine correlations between environmental factors and plant operational parameters over the overgrowth of foaming bacteria were utilized in this study. The results found Gordonia amarae was sequenced and was identified as a major foaming bacterial group. Our results showed a strong correlation between low temperature with high G. amarae cell population (r = -0.56 and P = 0.0011). Additionally, G. amarae cells increased when nitrification was not completed, and there was NO−2-N accumulation in the system and in second effluent (r = 0.68, P = 0.00; r = 0.46, P = 0.01) as well as elevated pH since nitrification was interrupted. Both findings implied low temperature associated with incomplete nitrification and nitrite accumulation were favorable factors to G. amarae’s growth compared to other bacteria.
Poster #: 266
Campus: CSU Fresno
Poster Category: Other
Keywords: beach management, biodiversity, bird surveys
Project Title: The effects of dredge deposition on avian and beach macroinvertebrate biodiversity in Morro Bay, California
Author List:
Moshier, Shelby; Graduate, Biology, California State University, Fresno, Presenting Author
Reece, Joshua; Faculty, Biology, California State University, Fresno
Abstract: Sandy beaches comprise the majority of coastlines globally and support diverse but narrow intertidal ecosystems. Humans are increasingly inhabiting coastal regions and exposing these ecosystems to anthropogenic activities. Morro Bay, located within San Luis Obispo County on California’s central coast, is home to sandy beach and estuarine ecosystems in addition to an economically valuable fishing industry and natural protected harbor. In February 2017 the harbor’s navigational channel was dredged to remove accumulated sediment, which was pumped approximately a mile upcoast as beach nourishment. While local-scale processes (i.e. dredging and beach nourishment projects) have been generally shown to impact beach ecosystems, we address the extent of these impacts specifically on Morro Bay beach biodiversity. In July 2017 we initiated a year-long study involving monthly bird counts and quarterly invertebrate counts at seven survey sites, including the deposition site, along the Morro Bay coast. From this census data, we calculate quantitative biodiversity values as determined by Simpson’s and Shannon’s diversity indices to evaluate spatiotemporal patterns of biodiversity and any significant differences correlated with dredging. Statistical tests show that while there is spatial and temporal variation in biodiversity between and across sites and months, deposition of the dredged material does not significantly decrease diversity at the site of deposition compared to the control sites. Our study will provide a greater understanding of the effects of dredging, which can help inform future decisions regarding potentially ecologically impactful projects in Morro Bay and elsewhere along the California coast. We would like to thank COAST and California State University, Fresno for providing funding for this project.
Poster #: 267
Campus: CSU Long Beach
Poster Category: Other
Keywords: Hippocampus, Sexual differentiation, Androgen receptor
Project Title: The Role of Androgen Receptor in Regulation of Sexual Dimorphism in Hippocampal Morphology of Juvenile Mice
Author List:
Song, Cherng-En; Undergraduate, Department of Biological Sciences, California State University, Long Beach, Presenting Author
Balbuena, Zuleica; Undergraduate, Department of Biological Sciences, California State University, Long Beach
Kim, Edward; Graduate, Department of Biological Sciences, California State University, Long Beach
Tsai, Houng-Wei; Faculty, Department of Biological Sciences, California State University, Long Beach, Presenting Author
Abstract: The hippocampus, in particular, is highly involved in spatial memory and processing navigational information, which has been long found to be sexually dimorphic in mice. Sex differences in spatial navigation, correlating with hippocampal morphology, with the majority of studies in the CA1 or the dentate gyrus (DG), have been attributed to organizational effects of perinatal testosterone. Since the hippocampus is reported to contain substantial levels of androgen receptor (AR) during early development, it is possible that AR activation by perinatal rises in testosterone might mediate hippocampal masculinization, leading to sex differences in spatial ability and hippocampal morphology. To test this hypothesis, we collected the whole brains of wild-type male and female mouse pups as well as males with testicular feminization mutation (Tfm), lacking functional AR, at postnatal days 21-24 (n=5-6 per group). Their brains were post-fixed in 4% paraformaldehyde, sectioned on a vibratome at the thickness of 25 µm, and stained with cresyl violet. After cover-slipping, measurement of thickness and area size of pyramidal cell layers in the CA1 and CA3, and granular cell layer in the DG of the hippocampus were taken under a light microscope using ImageJ. We found that the pyramidal cell layer of the CA3 in wild-type males (111.92 ± 10.83 µm) was thicker than wild-type females (85.04 ± 4.86 µm), and Tfm males (86.08 ± 4.28 µm) had a similar CA3 thickness of wt females (p=0.031). Interestingly, there was a trend of AR-regulated, the sex difference in area size of the CA3 pyramidal cell layer among wild-type and Tfm mice (p=0.054). In contrast to the CA3, none of these two stereological measurements in the CA1 or DG was different among the three groups of mice. Overall, our preliminary data support the essential role of AR in mediating the effect of perinatal androgens on creating sexual dimorphism in mouse CA3 pyramidal cell morphology during early development. Structural sex differences in CA3 pyramidal cells of juvenile mice might reflect fundamental differences in hippocampal function between the sexes.
Poster #: 268
Campus: CSU San Marcos
Poster Category: Other
Keywords: maternal, reward, conditioned place preference
Project Title: Modifications to Conditioned Place Preference Apparatus to Measure Maternal Motivation in Lactating Dams
Author List:
Torres, Jhair ; Staff, Psychology, California State University San Marcos, Presenting Author
D’Anna-Hernandez, Kimberly; Faculty, Psychology, California State University San Marcos, Presenting Author
Abstract: Reward processes related to pups in lactating dams support maternal behavior, but the underlying neural mechanisms of this pathway in the postpartum period are not well understood. Conditioned place preference (CPP) is a behavioral test that measures a preference to an area after pairing it with a rewarding stimuli. Past studies have used food or drugs to elicit a reward response in a chamber even if they weren’t present because the subjects learned to associate the chamber with them. Our goal was to alter CPP so it would be relevant for postpartum dams using pups as a reinforcer, as pups are a rewarding stimulus for dams. In this study, 13 postpartum mice underwent CPP. The apparatus consisted of three chambers. The two side chambers had either horizontal or vertical black and white stripes on the walls as visual stimuli and either corn cob or wood chip shavings as tactile cues on the floor. Dams were deprived of her pups for 90 minutes before conditioning. During conditioning, dams were placed in one chamber per day for one hour with or without her pups alternating chambers each day for four days postnatal days (PND) 3-6. On test day PND 7, the dam was allowed access to every chamber to test for preference. The results showed 7 of the 13 dams showed an increase in preference to the pup-associated chamber compared to baseline. Thus manipulating pup deprivation and exposure can elicit a place preference in dams and CPP may be a useful measure to study the underlying neuromechanisms of reward in the postpartum period. Experiments using the newly modified CPP are ongoing to determine the role of stress and arousal peptides in maternal motivation in mice.
Poster #: 269
Campus: CSU Long Beach
Poster Category: Product-focused Innovation
Keywords: diabetes, biomedical, innovation
Project Title: Development of a Handheld Medical Device for Saliva Screening
Author List:
Karwa, Talha; Undergraduate, Electrical Engineering, California State University, Long Beach, Presenting Author, Nagel Award Nominee
Bernal, Marisol; Undergraduate, Chemical Engineering, California State University, Long Beach
Milo, Austin; Undergraduate, Chemical Engineering, California State University, Long Beach
Tohmeh, Annas; Undergraduate, Electrical Engineering, California State University, Long Beach
Abstract: Due to a lack of affordable healthcare in poverty-stricken countries globally, the goal of this research is to provide low-cost and accessible methods of detection for diseases such as Type II Diabetes. From current development, the prototype biosensor works to detect when a light source is present, and the next step is to test the saliva reaction with the biosensor.
Human salivary amylase is an enzyme that catalyzes the hydrolysis of starch into simpler sugars. There has been a correlation reported between patients with diabetes and an increase in amylase levels. To measure their concentration of salivary amylase, we measured the light intensity of a fluorescent molecule that is produced in the reaction between salivary amylase and starch.
The biosensor’s purpose is to detect fluorescent molecules in a solution by measuring the intensity of light emitted by the molecules. The biosensor consists of a photodiode to measure the light at a certain wavelength and a transimpedance amplifier to convert the current signal into an amplified voltage signal. The program used for analysis of the incoming data is MATLAB.
Currently, we have ran the reaction using UV-vis spectroscopy to collect preliminary data as a proof of concept. This data consists of measuring the absorbance versus time at different concentrations of salivary amylase. It has been observed that as the concentration of salivary amylase increases, a faster steady state of the reaction is achieved. For the biosensor, tests have been done using different concentrations of BODIPY. Current tests show that the sensor is able to detect the fluorescence, but as the concentrations are closer together the photodiode is not able to recognize the differences due to a significant amount of noise. To counteract this noise, a digital filter using the Butterworth method was added to the MATLAB code, and this significantly improved the quality of the output signal.
Future work includes refining the MATLAB code and developing a dispensing and centrifuge system within the device. Furthermore, tests are being conducted using different fluorescent techniques with starch and salivary amylase. The overall goal of this research is to produce an affordable handheld device to detect Type II Diabetes as well as other diseases in the future. The completed handheld device will help make diagnosing diseases a reachable option for people living in low income areas around the world.
Poster #: 270
Campus: Maritime Academy
Poster Category: Product-focused Innovation
Keywords: Prosethetics, Grip-Strength, Control
Project Title: Development of An Affordable Pneumatic Hand Prosthesis and Control System
Author List:
Schorger, Kaelan; Graduate, Mechanical Engineering, California Maritime Academy, Presenting Author
Oppenheim, Tomas; Faculty, Mechanical Engieering, California Maritime Academy, Presenting Author
Abstract: Economic considerations and grip strength are significant limiting factors in the accessibility of prosthetic hands. Often, obtaining a myoelectric hand prosthesis capable of dealing with the demands of simple tasks comes with prohibitive costs, and often fall short of the grip strength required to deal with many tasks of everyday living. We have developed a novel high-grip and finger-pull-strength prosthetic hand is fabricated using “off the shelf” components. The prototype system we have developed is primarily focused on providing assistive functionality and accessibility to the user at an affordable cost. The prosthetic hand has three fingers, each actuated by pneumatic actuators, and it is controlled by myoelectric sensors. In addition, a novel three simultaneous degree of freedom (DOF) control scheme is used with independently developed software for control setup and user calibration. Each finger contains two joints, each capable of one degree of rotational freedom. The hand is capable of a power grip strength of 82 N, an individual fingertip grasping force of 52 N, and a 210 N fingertip holding force. The power grasp strength of the hand is above that of many commercial prostheses. While commercial robotic hand prostheses commonly cost tens of thousands of dollars for the hardware alone, our prototype system has been produced at a cost below $700.
In addition to the quantitative empirical testing described above, a variety of qualitative functional testing was conducted using the hand. This testing involved basic manipulation of a range of everyday objects, such as hand tools and food packages, as well as simple tasks of putting away a frying pan or lifting a barbell weight. In this testing of the hand, it was found to be well-suited to these common tasks, including those that required a relatively high degree of grip strength.
Poster #: 271
Campus: San José State University
Poster Category: Product-focused Innovation
Keywords: CRISPR, gene therapy, blood
Project Title: The design and validation of CRISPR guide RNAs specific for the RhD locus
Author List:
Kang, Sukhjiwan S.; Undergraduate, Biomedical Engineering, San José State University, Presenting Author
Swan, Chyna M.; Graduate, Biological Sciences, San José State University, Presenting Author
Kim, Enoch; Graduate, Biological Sciences, San José State University
Johnston, Jennifer M.; Faculty, Biological Sciences, San José State University
Abstract: The CRISPR-Cas9 nuclease platform is a powerful alternative to the limitations of viral-mediated gene therapy strategies. This platform can be employed to target clinically relevant genes to almost any active loci by exploiting the endogenous DNA repair mechanism of homologous recombination. However, prior to the addition of a therapeutic gene, a location capable of providing safe and sufficient expression must first be identified. Genomic safe harbors are inert locations within the genome able to permit the sufficient expression of exogenous genes without negatively impacting the host. Previously investigated safe harbors have since demonstrated deleterious effects as a result of gene addition. We are proposing the active RhD locus as a novel safe harbor for the integration of therapeutic transgenes for the treatment of monogenic diseases. This locus is a promising alternative, considering up to 43% of a population are classified as Rh-negative, and exhibit no phenotypic consequences as a result of disruption of the RhD gene. Thus, we designed seven 20 nucleotide CRISPR guide RNAs (gRNAs) to complement near the start codon. Our gRNAs were first designed manually, then confirmed using the algorithm based software CRISPRscan. Of these seven gRNAs, five complement on the sense strand and two complement on the antisense strand. These guides were subsequently incorporated into a Cas9 expression plasmid. Integration was confirmed by a restriction digest and sequencing. Targeting efficiency as evidenced by the presence of insertions and deletions was evaluated by both a TIDE and T7 Endonuclease I (T7EI) assay. Genomic DNA surrounding the gRNA target site from both CRISPR treated and non-treated cells was amplified using optimized PCR conditions. The PCR products were sequenced for TIDE analysis, and heteroduplex mismatches were generated at the site of indels for the T7EI assay. Preliminary TIDE analysis yielded a range of 5-60% cutting efficiency. The T7EI assay further confirmed these cutting efficiencies. Our most effective gRNA will subsequently be used to demonstrate integration and expression of an exogenous gene upon delivery with our DNA donor template, designed to facilitate homologous recombination. If successful, these experiments will serve as proof of principle for the future targeting of the RhD locus in hematopoietic stem and progenitor cells.
Poster #: 272
Campus: CSU Fullerton
Poster Category: Programmatic (Core, Stem Cell, Bridges, PSM)
Keywords: lower division undergraduate research, cross-disciplinary program, DNA origami
Project Title: Empowering Lower Division Undergraduates to Engage in Cross-Disciplinary Research
Author List:
Arroyo, Daniel; Undergraduate, Biology, California State University, Fullerton, Presenting Author
Chen, Binyun; Graduate, Mechanical Engineering, California State University, Fullerton, Presenting Author
Rasche, Madeline; Faculty, Biochemistry, California State University, Fullerton
Robson, Nina; Faculty, Mechanical engineering, California State University, Fullerton
Abstract: Active participation in undergraduate research has been shown to play a valuable role in enhancing the educational experience of undergraduate science and engineering majors. In addition to endorsing learner-centered approaches and participation in discovery-driven research, the National Research Council (NRC) promotes the development of genuinely interdisciplinary courses and curricula, leading to scientists and engineers capable of combining specialized disciplinary knowledge with fluency in complementary disciplines. This vision is consistent with a recent report of the California Life Sciences Institute (CLSI) that workforce-ready biotechnology candidates tend to be equipped with soft skills, as well as experience in using modern data-driven approaches in science, such as robotics, bioengineering, computational modeling.
The authors have combined the NRC with the CLSI calls, by integrating research-based high-impact practices into a lower division undergraduate cross-disciplinary program on Early Experiences in Bioengineering/Biotechnology in the area of DNA Origami. The program involves students from the Colleges of Natural Science and Engineering and Computer Science and spans the Spring semester during freshmen, a summer internship and the Fall semester during sophomore year. The curriculum activities are designed to strengthen the lower division foundation for authentic cross-disciplinary undergraduate research productivity and preparation for jobs in the biotechnology workforce.
Our preliminary direct pre- and post-course self assessment survey results show that for the short time of one semester and a month of summer internship, the program enhanced the students’ (i) readiness for cross-disciplinary research by 32%, (ii) preparation for upper division research by 25%, (iii) acquisition of laboratory problem solving and scientific communication skills by 28%, and (iv) scientific research literacy by 32%. The survey also reveals that the engineering and the biological concepts were equally difficult for the students spanning both colleges, which means that the course activities and projects were well balanced. On average, the students shared that the biological concepts were slightly more difficult than the mechanical engineering concepts (65% v/s 62.5%). The last semester of the program involves improving the students’ preparation for upper division research and exposing them to career opportunities in bioengineering and biotechnology.
Poster #: 273
Campus: CSU Fullerton
Poster Category: Proteins (Include Proteomics)
Keywords: PTBP1, nucleocytoplasmic shuttling, immunofluorescence
Project Title: A Potential Secondary Function of One of the Four RNA Binding Domains in PTBP1
Author List:
Peralta, Dulce; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Miyamoto, Alison; Faculty, Biological Science, California State University, Fullerton
Keppetipola, Niroshika; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Abstract: Alternative splicing is an important mechanism that increases protein diversity and contributes in the modulation of gene expression. Polypyrimidine Tract Binding Protein 1 (PTBP 1) is an RNA binding protein that contains four RRM-type RNA binding domains connected by three linker regions and an N-terminal region which contains a nuclear export sequence (NES) and a nuclear localization sequence (NLS). In the nucleus, PTBP 1 plays a role in the regulation of alternative splicing and polyadenylation. In the cytoplasm, PTBP1 functions in mRNA localization, stability and translation (Keppetipola et at al 2012). Thus, to carry out its nuclear-cytoplasmic functions PTBP1 shuttles between the two cellular compartments. Previous studies highlight that in addition to the classic NES signal, PTBP1 nuclear export is also regulated by the RRM2 domain; deletion of this domain inhibited nuclear export of PTBP1 (Kamath et al. 2001). Recently several lysine residues in the RRM2 domain were discovered to be post translationally acetylated (Pina et al. 2018). Thus, we hypothesized that post-translational acetylation regulates PTBP1 nuclear export. To test this, we conducted an alanine scan of four acetylated lysine residues (K212A, K218A, K259A, and K266A) in the RRM2 region and assayed the mutants along with wild type PTBP1 for cellular localization via immunocytochemistry. Wild type PTBP1 and mutants were FLAG-tagged. These studies were performed in two different cell lines (osteosarcoma MG-63 and human embryonic kidney cells 293T), with anti-FLAG antibodies to detect the PTBP1 proteins and DAPI to detect the nucleus. As reported previously our results highlight that at steady state wild type PTBP1 is predominantly nuclear with some cytoplasmic localization (Kamath et al. 2001, Li et al. 2002, Xie et al. 2003). Mutants K218A and K259A localized primarily in the nucleus suggesting these residues do not play a role in PTBP1 localization. In contrast, mutants K266A and K212A localized primarily in the cytoplasm. This suggests a role for Lys266 and Lys212 in PTBP1 nuclear export, specifically that acetylation at these two positions modulate nuclear export. To our knowledge this is the first evidence that PTBP1 localization is regulated by lysine acetylation in the RRM2 domain. Funding from CIRM Bridges 2.0 (DP) and CSUF Research Scholarly and Creative Activities Award (NK).
Poster #: 274
Campus: CSU Los Angeles
Poster Category: Proteins (Include Proteomics)
Keywords: recombinant protein, Escherichia coli, expression optimization
Project Title: Optimization of Recombinant a Beetle Antifreeze Protein Production in Escherichia coli
Author List:
Lee, Kimberly; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Lu , Cathy; Temple City High School, Presenting Author
Eneque, Belen ; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles
Lomibao , Roxanne ; East Los Angeles College
Ortiz, Jocelyn ; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles
Salgado, Rubi ; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles
Perez , Stephany ; Alliance Marc & Eva Stern Math and Science School
Lopez , Daveed ; East Los Angeles College
Wen, Xin; Faculty, Chemistry and Biochemistry, California State University, Los Angeles
Abstract: Escherichia coli, a gram-negative bacterium, has been an attractive host system for heterologous protein production. There are many advantages of using E. coli to produce recombinant proteins, such as inexpensive and rapid growth and access to various cloning vectors. The production of eukaryotic proteins in E. coli, however, may not yield high production. Factors including bacterial growth temperatures, expression conditions, and growth media need to be optimized to achieve high yield of heterologous proteins overproduction in E. coli. Antifreeze proteins (AFPs) have been proven to play a crucial role in subzero organism’s survival by inhibiting the formation of ice crystals in low temperatures. The relevance between AFPs and their commercial applications has been an area of interest for pharmaceutical companies, cryopreservation, and the food industry. In this study, we grew a recombinant AFP from Dendroides canadensis (DAFP) in E. coli in three different media, Luria Bertani broth (LB), Plasmid+, and terrific broth (TB) media. The bacterial growth temperatures and expression conditions were controlled during all the growths. The expressions of the recombinant AFP in different media were monitored using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results show that little AFP was expressed in the E. coli grown in Plasmid+ medium, while the optimal expression of the AFP was achieved in the E. coli grown in TB. The cells grown in LB and TB were then collected for protein purification and the protein production in TB and LB were compared. Our results suggest that optimizing culture medium can enhance the production of recombinant AFPs in E. coli. The experimentation and diversification in uses of AFPs have suggested a higher demand in AFPs production. The optimizations presented in this study allow the production of these useful heterologous proteins at a higher yield and rate.
Poster #: 275
Campus: CSU Long Beach
Poster Category: Proteins (Include Proteomics)
Keywords: Sexual Differentiation, Brain, RNA binding protein
Project Title: Sexually Dimorphic Expression of RBM48 Protein in the Developing Mouse Cerebral Cortex and Hippocampus
Author List:
Lertpanit, Long; Graduate, Department of Biological Sciences, California State University, Long Beach, Presenting Author
Tsai, Houng-Wei; Faculty, Department of Biological Sciences, California State University, Long Beach, Presenting Author
Abstract: Sexual differentiation influences sex-specific development and function of many brain regions, including the cerebral cortex and hippocampus. These two brain regions are known to play an important role in physiological processes linked to complex social and cognitive behaviors, many of which are sexually dimorphic. However, the molecular mechanisms underlying these sex differences are only beginning to emerge. Our lab has previously found higher expression of RNA binding motif protein 48 (Rbm48) mRNA in the cortex/hippocampus of female mice than males at postnatal days (PN) 0 and 7. Due to the presence of an RNA recognition motif, known as RNA-binding domain (RBD), the Rbm48 gene encoding protein is speculated to be involved in RNA biogenesis, stability, function, transport, and cellular localization. Based on these findings, we propose Rbm48 might function as an integrator of hormonal information to modulate brain sexual differentiation at the posttranscriptional level. Prior to investigating its molecular and neural function, we wanted to determine if the sex-based difference in protein levels of RBM48 was similar to that observed on the mRNA level. To test this, we used immunoblotting to measure relative RBM48 protein levels in the male and female mouse cerebral cortex/ hippocampus collected at PN7 (n=6 for each sex). Proteins extracted from those cortex/hippocampus samples were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, followed by transfer to nitrocellulose membranes. After blocking, membranes were immunoblotted for RBM48 and then β-actin (as loading control). RBM48 and β-actin protein bands were detected at ~50 and 42 kDa by chemiluminescence and the FluorChem E Imaging System (ProteinSimple, Santa Clara, CA). The ratios of RBM48 and β-actin band intensities were normalized to the mean of females (as one-fold). A two-sample t-test revealed that female mice showed higher RBM48 expression in the cortex/hippocampus than males (1.00 ± 0.14 vs. 0.53 ± 0.08 fold; p=0.023). We are currently verifying if the same sex difference in RBM48 protein levels of the cortex/hippocampus also exists in neonatal and older mice. Our preliminary results not only support that RBM48 is differentially expressed in the mouse cortex/hippocampus between the sexes at mRNA and protein levels during early development, but also lay a foundation for further investigation of the molecular functions of RBM48 in brain sexual differentiation.
Poster #: 276
Campus: CSU Sacramento
Poster Category: Proteins (Include Proteomics)
Keywords: apolipoprotein, amyloid, protein folding
Project Title: Assessing the effect of heparin on acid-induced aggregation in apolipoprotein A-I variants
Author List:
Nguyen, Nancy; Graduate, Chemistry, California State University, Sacramento, Presenting Author
Roberts, Linda; Faculty, Chemistry, California State University, Sacramento, Presenting Author
Abstract: Apolipoprotein A-I (apoA-I), a major component of high density lipoprotein (HDL), is strongly associated with lowered risk for heart disease. Despite its anti-atherogenic properties, apoA-I deposits as amyloid in various tissues and organs, including the heart. ApoA-I forms amyloid either by mutation or influence of environmental factors during physiological processes. We and others have shown that acidic pH induces non-specific aggregation. Townsend and coworkers further demonstrated that heparin stimulates formation of amyloid structure at acidic pH, likely through electrostatic interactions. The effect of heparin on acid-induced aggregation of amyloidogenic mutants has not been investigated. The goal of this research is to compare the effect of acid pH with and without heparin on aggregation in full-length apoA-I and two of its amyloidogenic mutants, G26R and L178H. Presented here are studies extending the exploration of acidic pH on WT apoA-I and preliminary examination of G26R. Purified apoA-I protein was combined with Mcllvaine’s buffer in the presence and absence of heparin to a final protein and heparin concentration of 0.1 mg/mL. Samples were incubated at 37 degrees Celsius for time intervals of 1, 24, 48 and 72 hours with agitation. Aggregation of protein was determined using light scattering intensity at 340 nm while amyloid formation was detected by Thioflavin T (ThT) fluorescence. Intrinsic and ANS fluorescence was used to detect conformational changes. Our findings show that both full-length wild-type and mutant G26R aggregate extensively at pH 4.0, 4.5 and 5.0 in the presence and absence of heparin. Intrinsic fluorescence of WT increased in the presence of heparin and was blue-shifted at pH 4 and 4.5, indicating a more nonpolar environment of the protein’s four tryptophan residues. ThT fluorescence was much higher in WT samples containing heparin at pH 4.0-5.0 and appeared in pH 5.5 and 6.0 samples after 48 and 72 hours, respectively, indicating increased formation of amyloid structure over time at increased pH. ANS fluorescence was higher at pH 4.0, 4.5 and 5.0 in WT apoA-I samples and was blue-shifted in the presence of heparin, indicating a structural change due to heparin binding. These results indicate that WT and G26R protein have similar responses to acidic pH and that heparin accelerates the formation of amyloid structure WT apoA-I which may contribute to the formation of amyloid in acidic microenvironments in vivo.
Poster #: 277
Campus: San Diego State University
Poster Category: Proteins (Include Proteomics)
Keywords: protein cleavage, classical secretory pathway, cell surface
Project Title: Development of a platform that monitors cleavage during transport to/and at the cell surface
Author List:
Ventola, Francesca; Graduate, Biology, San Diego State University, Presenting Author
Salazar, Sergio; Undergraduate, Biology, San Diego State University
Nader, Joy; Graduate, Biology, San Diego State University
Smurthwaite, Cameron; Staff, Biology, San Diego State University
Wolkowicz, Roland; Faculty, Biology, San Diego State University
Abstract: Many viral proteins are processed by host proteases as they travel through the classical secretory pathway of the cell, involving the Endoplasmic Reticulum/Golgi/Trans-Golgi network. This is exemplified by the HIV envelope (Env) protein and the pre-membrane protein of Dengue Virus. Other proteins are rather cleaved at the cell surface, further proving the importance of the secretory pathway for biological processes. We have thus embarked in a biotechnological approach to monitor cleavage within this pathway in both locations: inside the vesicles as proteins travel to the surface, and at the cell surface itself. We first developed an assay that can monitor Env cleavage in an attempt to find new antiviral targets. The Env polyprotein gp160 is processed into two subunits; gp120 and gp41, by the host protein convertase Furin, within the Golgi apparatus in its way to the cell surface. To monitor cleavage, we engineered a 17 amino-acids portion of the gp120/41 boundary embedded within a scaffold protein flanked by HA and FLAG tags that travels through the ER/Golgi/TGN. On its way to the cell surface, the FLAG tag is removed by Furin. Through antibody staining and fluorescent-based detection by microscopy and flow cytometry, we monitor whether Env was cleaved or not based on the presence of one or two tags at the cell surface. We have further developed the two-tag system that monitors cleavage intracellularly for the monitoring of proteolytic events that specifically occur at the cell surface or extracellular matrix (ECM). Proteolysis at the ECM plays a significant role in cancer progression, metastasis, and other diseases. This is exemplified by Matrix Metalloproteinase 14 (MMP-14). For that reason we have replaced the HIV Env boundary with an optimized MMP-14 substrate. In order to prove cleavage by MMP-14 we concomitantly expressed the MMP-14 enzyme. Flow cytometry data show a dramatic reduction in FLAG expressing cells occurs only in the presence of MMP-14. To further prove cleavage at the cell surface we have performed mixing experiments with two separate populations of cells expressing either MMP-14 or the substrate. By mixing substrate-expressing cells with increasing MMP-14-expressing cells, we observed a reduction of the FLAG level expression, demonstrating cleavage at the cell surface. Confocal microscopy further proves substrate and enzyme cell surface expression. We are currently adapting both assays to obtain a high-throughput platform for screening.
Poster #: 278
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: biophysics, nucleic acids, fluorescence
Project Title: Synthesis and Photophysical Properties of Modified Nucleic Acid Oligonucleotides with Fluorescent Tricyclic Cytidine
Author List:
Turner, Marc; Graduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Ceja, Jesús; Graduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Coste, Michael; Graduate, Department of Chemistry and Biochemistry, San Diego State University
Purse, Byron; Faculty, Department of Chemistry and Biochemistry, San Diego State University
Abstract: We have developed new chemically modified DNA probes that can increase their fluorescence when annealed to complementary DNA/RNA sequences. Fluorescence-based detection of specific DNA/RNA sequences using classic fluorescence in situ hybridization (FISH) has been crucial in optically detecting specific nucleic acids in biological samples. Common targets may include specific mRNA, plasmid DNA, or the 16S rRNA of potential bacterial pathogens. Fluorescent probes frequently, however, yield false positives by hybridizing to similar sequence targets differing little in the thermodynamic stability of the duplex. Probes with our lab’s tricyclic cytidine derivatives can potentially discriminate sequences more accurately than current hybridization methods. In our lab’s previous work, the tC derivate, 8-diethylamino-tC (8-DEA-tC), was shown to have a fluorescence turn-on property. This analogue has low intrinsic emission of fluorescence (Φem = 0.01), however the emission can increase up to Φem = 0.12 when 8-DEA-tC pairs with guanosine. In this work we measure the photophysical properties of 8-DEA-tC DNA-RNA heteroduplexes, which have resulted in larger increases in 8-DEA-tC fluorescence. When 8-DEA-tC is flanked by guanosine on the 5′ side and cytosine on the 3′ side, the emission ratio, as measured using a QuantaMaster fluorometer, increases from 0.65% to 9.4%, resulting in a 14-fold increase. 8-DEA-tC may be especially useful for recognizing specific RNA in biological samples. We are also developing tC-derived peptide nucleic acids (PNA) probes as alternative fluorescence reagents since their modified structure confers higher binding affinities to DNA or RNA targets. The synthesis of parent tC PNA Boc-protected monomer has been completed with an approximate overall 35% yield over four steps. Synthesis of the 8-DEA-tC PNA monomer is currently in progress. After solid-phase peptide synthesis, the tC-derived PNA probes will be characterized by fluorescence spectroscopy and circular dichroism to measure their photophysical properties. Developing more accurate techniques for detecting specific sequences is highly informative in biomedical research and biotechnology.
Poster #: 279
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: antibiotics, medicinal chemistry, organic synthesis
Project Title: A New Hope For Antibiotic Drug Discovery: Nucleoside analogue inhibitors of GTP cyclohydrolase I (GCYH-I) as a potential new class of antibiotics
Author List:
Samaan, George; Graduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Wyllie, McKenzie; Undergraduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Paranagama, Naduni; Graduate, Department of Chemistry and Biochemistry, San Diego State University
Swairjo, Manal; Faculty, Department of Chemistry and Biochemistry, San Diego State University
Purse, Byron; Faculty, Department of Chemistry and Biochemistry, San Diego State University
Abstract: Despite a pressing global need, no new class of antibiotics has been clinically approved in more than 30 years. This project seeks to develop a new class of antibiotic drugs to be used against resistant pathogens such as MRSA. Our goal is to design and synthesize novel antifolate compounds, which will inhibit the synthesis of folate in bacteria, leading to bacterial death. The enzyme GTP cyclohydrolase I catalyzes the first step in folate biosynthesis in bacteria and biopterin biosynthesis in humans. Many bacteria use an essential GTP cyclohydrolase I enzyme, GCYH-IB, that bears little structural homology to the human form, GCYH-IA. The crystal structures of GCYH-IA and -IB reveal that the active site of GCYH-IB is significantly larger, suggesting that it can be inhibited selectively by molecules too large for binding to GCYH-IA. Our team has found that a known substrate-analogue inhibitor of GCYH-IA, 8-oxo-GTP, inhibits also bacterial GCYH-IB, but with reduced potency. By modifying 8-oxo-GTP to exploit active site features unique to GCYH-IB, we propose to develop new inhibitors with high selectivity for the bacterial enzyme. We have designed and synthesized an initial set of compounds to test structure–activity relationships in the inhibition of GCYH-IA and -IB. Two compounds, G1 and G2, showed lower inhibition of the bacterial enzyme than of the human enzyme by in vitro assays of product formation. The third compound (S)-G3 showed no significant difference in the inhibition of the two enzymes. A further modification, G3, showed more inhibition of the bacterial enzyme (50%) than the human enzyme (15%), demonstrating in principle that these active site differences can be targeted. The G3 compound is unique because it binds more to the bacterial than the human enzyme, thereby reversing the selectivity of 8-oxo-GTP. We are developing synthetic strategies using various reactions to add different functional groups to G3, modifying it to create an optimum inhibitor to the bacterial enzyme. Our future is to test the modified inhibitors against the target bacterial cells (e.g. Neisseria gonorrhoeae and Staphylococcus aureus) and make prodrugs that facilitate the penetration of the inhibitors into the bacterial cell membrane.
Poster #: 280
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: atropisomers, kinase inhibitors, optimization
Project Title: Structural Optimization of Atropisomeric Pyrrolopyrimidine Ret Kinase Inhibitors
Author List:
Toenjes, Sean; Graduate, Chemistry & Biochemistry, San Diego State University, Presenting Author
Garcia, Valeria; Undergraduate, Chemistry & Biochemistry, San Diego State University, Presenting Author
Maddox, Sean; Postdoc, Chemistry & Biochemistry, San Diego State University
Dawson, Gregory; Graduate, Chemistry & Biochemistry, San Diego State University
Cardenas, Mariel; Graduate, Chemistry & Biochemistry, San Diego State University
Saputra, Della; Postdoc, Chemistry & Biochemistry, San Diego State University
Gustafson, Jeffrey; Faculty, Chemistry & Biochemistry, San Diego State University
Abstract: There are over 500 kinase proteins in the human genome, and their aberrant activity can lead to
life-threating diseases. Consequently, a dense amount of medicinal research has been focused on the
development of small molecule kinase inhibitors. More than 80% of the FDA approved kinase
inhibitors contain rapid interconverting chirality, also known as atropisomerism, and although they
are considered to be achiral, they interact with their targeted proteins in an enantioselective fashion. This means that while one enantiomer contributes towards the desired activity, the other enantiomer can inhibit off-target proteins which may lead to adverse side effects in patients or muddled biochemical signaling pathway experiments.
In research done previously in the Gustafson lab, exploiting atropisomerism was used as a
selectivity filter and to increase the selectivity of pyrrolopyrimidine (PPY) Ret kinase inhibitors. In their report, they rigidified a bi-aryl axis by adding bulky substituents ortho to the chiral axis and observed that the (Ra)-atropisomer was 2-fold more selective towards Ret over Src. One drawback of this research was by rigidifying the chiral axis resulted in the loss of potency compared to the parent racemizing inhibitor (1857nM vs 128nM IC50 towards Ret).
To further demonstrate that this strategy of exploiting atropisomerism as a selectivity filter we must optimize the inhibitors for both selectivity and potency. To do this, we utilized a molecular docking software called MOE to create a list of potential analogues that were calculated to be both selective and potent for Ret over Src. We then synthesized the molecules (15 scaffolds, 28 atropisomers) and separated the (Ra)- and (Sa)-atropisomers through chiral phase high liquid performance chromatography (HPLC). These isolable, stable atropisomers were then subjected to kinase inhibition assays to test the inhibition of the molecules. Structure activity relationships (SAR) showed a larger ‘gate keeper’ aryl ring and an electron neutral methyl group off the PPY increased potency towards Ret (17nM IC50) and selectivity (>100-fold Ret/Src and >500-fold Ret/Vegfr2). The lead inhibitor displayed cytotoxicity (IC50 2.5uM) in estrogen-deprived breast cancer cells (ED-MCF7) and showed elimination of Ret phosphorylation via western blots. Both the in silico and in vitro data suggest that we can obtain a higher potency while maintaining the selectivity in novel atropisomeric inhibitors.
Poster #: 281
Campus: CSU San Marcos
Poster Category: Synthetic Chemistry
Keywords: organocatalysis, synthesis, enantioselective
Project Title: Chiral Bisphosphorylimides as Organocatalysts for Enantioselective Friedel–Crafts Reactions
Author List:
Apolinar, Omar; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author, Nagel Award Finalist
Iafe, Robert; Faculty, Chemistry & Biochemistry, California State University San Marcos
Abstract: The importance of efficient enantioselective synthesis and purification increases as an growing number of chiral bioactive molecules flood the pharmaceutical industry. 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. 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. The Friedel–Crafts reaction is one of the most efficient methods for making new carbon-carbon bonds on benzene rings, and bisphosphorylimides are currently underutilized in this catalytic role. The carbon scaffold of bisphosphorylimides is highly 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 building blocks useful in the synthesis of chiral bioactive molecules.
Results: Two new bisphosphoylimide derivative (Ar = phenyl, anthracenyl) have been prepared in moderate yields using the Suzuki reaction from enantiopure BINOL. These catalysts were fully characterized by NMR and IR spectroscopy. Initial substrate scope investigation on three different benzylidenecarbamate substrates using optimized reaction conditions (5 mol % catalyst, 2.5 mol % base, 1.5 hours, reduced temperature) previously determined in our lab showed emerging enantioselective excess despite complete substrate conversion. Furthermore, our lab is developing novel methodology to prepare late-stage modifications of the bisphosphorylimide organocatalyst which involves performing a quadruple Suzuki reaction as the final step. Current literature preparation introduces the Suzuki reaction early in the synthesis, however this has deleterious effects in overall yield because phosphorylation is not very efficient. Late stage differentiation not only makes the process more cost-effective, but access to a library of modified catalysts can be prepared in less time.
Acknowledgements: We thank CSUPERB (New Investigator) and CSUSM Summer Scholars for support of this work.
Poster #: 282
Campus: CSU San Marcos
Poster Category: Synthetic Chemistry
Keywords: gold catalysis, benzotriazoles, methodology
Project Title: Microwave-Assisted Gold(I) Reaction of Benzylic Alcohols for the Preparation of Modified 1,2,3-Benzotriazoles
Author List:
Nguyen, Mercedes; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author, Nagel Award Nominee
Perez, Jacqueline; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author
Iafe, Robert; Faculty, Chemistry & Biochemistry, California State University San Marcos
Abstract: Applications of 1,2,3-benzotriazole derivatives and their syntheses have attracted significant interest within the past few decades. The 1,2,3-benzotriazole moiety is a ubiquitous structural constituent of corrosion inhibitors. This motif is also common in many biologically active compounds, of which have anticancer, antifungal, anti-inflammatory, antimicrobial, and cytotoxic activity. Alkylated derivatives of these molecules also serve as synthetic precursors for difficult nucleophilic reactions. During our prior investigation using gold(I) salts to catalyze etherification reactions, we discovered that 1,2,3-benzotriazoles have nucleophilic reactivity in preference to the etherification pathway. To our satisfaction, we were successfully able to cleanly promote the N-alkylation product using microwave conditions. From this, our lab has developed the first microwave-assisted, gold(I)-catalyzed substitution reaction of 1,2,3-benzotriazole with benzylic alcohols to afford N-substituted 1,2,3-benzotriazole derivatives. To our knowledge, while examples of base-catalyzed and phase transfer catalysis displacement reactions are well-known, a microwave assisted, gold(I)-catalyzed substitution 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 displacement reaction has been optimized using 1,2,3-benzotriazole as the nucleophile, 1-phenylethanol as the electrophile, 5 mol % of the gold(I) and silver(I) salts, and elevated temperatures to afford 99% of the target compound. Using these conditions, the substrate scope for this reaction was explored using electrophiles of various electronic and steric properties. Excellent yields (45–99%) were obtained when the electronics were varied on the electrophile; however, strongly electron withdrawing groups shut the reaction down. The gold(I)-catalyzed reaction was also used in the preparation of an anticancer drug (99% yield) to demonstrate pharmaceutical application. Also, comments on the regioselectivity of this reaction will be presented, as data supports this preference is mostly attributed to Van der Waals interactions.
Poster #: 283
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Synthesis, Anti-Cancer, Anti-Malarial
Project Title: A Highly Stereoselective Synthesis of Lagunamide A: A Metabolite from the Lyngbya Majuscula Cyanobacterium Possessing Powerful Anti-Cancer Properties
Author List:
Stillman, Jared; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Feese, Sydney; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Singh, Simranjeet; Staff, Chemistry and Biochemistry, San Diego State University
Wang, Lee; Staff, Chemistry and Biochemistry, San Diego State University
Bergdahl, Mike; Faculty, Chemistry and Biochemistry, San Diego State University
Abstract: Background: Lagunamide A is a natural product originally isolated from Cyanobacteria with an exceptional biological activity showing great promise as a future therapeutic agent. Lagunamide A has many biological activities and exceptional IC50 values. High cytotoxicity is present against P388 murine leukemia cell lines (IC50 6.4-20.5 nM) and Ileocecal colon cancer (IC50 1.6 nM). This substance has also impressive anti-malarial properties (IC50 0.19-0.91 micro-M). With such noteworthy activities, Lagunamide A shows great promise as a powerful therapeutic agent and brings the need for a shorter and higher yielding total synthesis of this macrocyclic depsipeptide, which would allow for easier biological studies. Current data shows the depsipeptide mode of action having an intrinsic apoptotic pathway by cleavage of capsase-9 which activates a series of cascades resulting in mitochondrial assisted apoptosis.
Results: We will present a novel stereocontrolled total synthesis of Lagunamide A. This encompasses a highly convergent asymmetric route that installs 5 of the 10 critical stereocenters. Four stereocenters are installed with high accuracy using two novel iterative Vinylogous Mukaiyama Aldol Reactions with high yields and excellent diastereomeric ratios. This is the highest yielding and shortest synthetic route for obtaining Lagunamide A. Although the VMAR methodology initially was a challenge for construction of the backbone, a systematic fundamental study was created and subsequently optimized, and these data applicable to the synthesis of Lagunamaide will be presented. In addition, we will present a new optimized method for coupling unique N-methylated unnatural peptide fragments completed via solid phase synthesis, which is the most convenient route for creating the northern hemisphere of Lagunamide A. All data for each intermediates presented have been fully characterized via NMR spectroscopy, X-ray crystallography, FTIR, HPLC, and LCMS. This sets the stage for future work of Lagunamide A to be the syntheses of analogs for structure-activity relationship (SAR) studies which will be tested against various malarial and cancer cell lines.
Poster #: 284
Campus: CSU Sacramento
Poster Category: Synthetic Chemistry
Keywords: dendrimer, anti-viral, synthesis
Project Title: Optimization of an Octavalent Dendrimer Core Synthesis
Author List:
Zeman, Kara; Undergraduate, Department of Chemistry, California State University, Sacramento, Presenting Author
Cerney, James; Graduate, Department of Chemistry, California State University, Sacramento
McReynolds, Katherine; Faculty, Department of Chemistry, California State University, Sacramento
Abstract: Dendrimers are macromolecules comprised of a core and branches that create a spherical shape with chosen functional groups on the surface, allowing for multivalency to occur. Multivalency is the property of multiple copies of one entity binding to multiple receptors on another. Dendrimers have a variety of biological applications because of this and can be manipulated in branch length, composition, and valency to increase stability, solubility, and therefore, usefulness. This research focuses on optimization of the synthesis of octavalent dendrimers, seeking greater efficiency, higher yields and improved purity. Synthesis of an octavalent dendrimer has been achieved via an eight-step divergent pathway. In the first step, t-butyl acrylate underwent a Michael addition with ethylene glycol (39.8% yield). The t-butyl linker was then modified with tosyl chloride (94.5% yield). The tosylated linker was next coupled with ¬¬¬¬¬2,2’oxybis(ethylamine) through a substitution reaction, followed by immediate deprotection to give the tetravalent carboxyl-terminated core (33.4%, two-step, one-pot yield). The four branches of the dendrimer were then further lengthened through a Fischer esterification with methanol, followed by amination (95.9%, two-step-one-pot yield), branching with t-butyl acrylate (32.6% yield), and then deprotection (7.7% yield), to give the octavalent carboxyl-terminated dendrimer. The error is amplified with multiple low yielding steps, so various synthetic tactics will be employed to improve these steps. Once optimized, future work will be done to add various sugars to the dendrimer core, creating glycodendrimers, which can then be evaluated for antiviral activity.
Poster #: 285
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Angiogenesis, anti-cancer, organic chemistry
Project Title: A simple total synthesis of (+)-Azaspirene, a formidable anti-angiogenesis instrument against cancer
Author List:
Black, Jourdan; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Bender, Anastasiya; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Walsworth, Kevin; Staff, Chemistry and Biochemistry, San Diego State University
Bergdahl, Mike; Faculty, Chemistry and Biochemistry, San Diego State University
Hecht, David; Faculty, Chemistry and Biochemistry, San Diego State University
Abstract: Background: (+)-Azaspirene, a natural substance isolated from the soil fungus Neosartorya sp., is a novel powerful and favorable anti-cancer agent that inhibits the creation of new blood vessels for growth of cancer cells via a process called angiogenesis. In a normal cell growth angiogenesis is also the instrumental creation of new blood vessels for growth and wound repair. Tumors reroute these natural functions to support their uncontrolled growth and azaspirene has been shown to disrupt the inordinate signaling pathway associated with tumor angiogenesis, thus stopping tumor growth without harming normal body functions. Due to its mechanism of action, azaspirene, unlike most modern cancer drugs, has also extremely low level of toxicity and has therefore a high potential for use as a synergistic form of chemotherapy. Currently, the supply of azaspirene from either natural sources or synthetic methods is too small for further biological research or drug use. As such, we developed an efficient synthetic route is to alleviate these challenges.
Results: Our results demonstrate an efficient asymmetric synthesis of (+)-azaspirene. This reported route is also designed to allow the access of pseurotin type hybride structures, a group of highly bio-active relatives to azaspirene. Scalable reactions and cheap starting materials have been used, which allows for easy access to these elusive molecules. This will also allow for in depth biological testing and potential for large scale synthesis. Since a sufficient quantity of azaspirene has been obtained it allows for the determination of azaspirene’s binding site and further evaluation of azaspirene as an anti-angiogenic and anti-tumor agent. Our synthesis is initiated with L-phenylalanine and has been completed in 11 steps and in a record 6% overall yield. Our results will be discussed and compared to the previously reported synthesis in over 30 steps to access azaspirene.
Poster #: 286
Campus: CSU San Marcos
Poster Category: Synthetic Chemistry
Keywords: small molecule synthesis, methodology, gold catalysis
Project Title: Preparation of Bioactive 1,2,3-Benzotriazole Targets by a Microwave-Assisted Gold(I) Catalysis Procedure
Author List:
Shabo, Mariana; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author, Nagel Award Nominee
Franco-Bolanos, Victor; Undergraduate, Chemistry & Biochemistry, California State University San Marcos
Iafe, Robert ; Faculty, Chemistry & Biochemistry, California State University San Marcos
Abstract: Applications of 1,2,3-benzotriazole derivatives and their syntheses have attracted significant interest within the past few decades. The 1,2,3-benzotriazole moiety is a ubiquitous structural constituent of corrosion inhibitors. This motif is also common in many biologically active compounds, of which have antifungal activity. 1,2,3-Triazole antifungals act by inhibiting the conversion of lanosterol 14-alpha-demethylase (CYP51) into ergosterol, the primary sterol in fungal cell membranes. During our prior investigation using gold(I) salts to catalyze etherification reactions, we discovered that 1,2,3-benzotriazoles have nucleophilic reactivity in preference to the etherification pathway. To our satisfaction, we were successfully able to cleanly promote the N-alkylation product using microwave conditions. From this, our lab has developed the first microwave-assisted, gold(I)-catalyzed substitution reaction of 1,2,3-benzotriazole with benzylic alcohols to afford N-substituted 1,2,3-benzotriazole derivatives. To our knowledge, while examples of base-catalyzed and phase transfer catalysis displacement reactions are well-known, a microwave assisted, gold(I)-catalyzed substitution 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 synthesis of two different bioactive compounds have been improved compared to literature using our gold(I) microwave-assisted protocol. (1) A 1,1-diarylmethane derivative with antifungal properties was synthesized in 87% over two steps from benzaldehyde, compared to a three-step protocol in 44% yield found in literature. (2) A ferrocenylalkyl 1,2,3-benzotriazole derivative with anticancer properties was also prepared in 98% yield over three steps from ferrocene. This synthetic route also avoids unwanted constitutional isomer byproducts that originate from substitution at the N2 position.
Acknowledgements: We thank CSUSM Summer Scholars for support of this work.
Poster #: 287
Campus: San José State University
Poster Category: Synthetic Chemistry
Keywords: alkane oxidation, inorganic catalysis, active-site mimics
Project Title: Enzyme-Like Catalysis: Encasing Methane Monooxygenase Mimics in a Polymer Scaffold for Improved Catalysis
Author List:
Ordona, Harris; Undergraduate, Chemistry, San José State University, Presenting Author
Tafuri, Victoria; Undergraduate, Chemistry, San José State University, Presenting Author, Nagel Award Nominee
Navarro, David; Undergraduate, Chemistry, San José State University
Abstract: An efficient conversion of methane – a potent greenhouse gas – to methanol – an energy-dense liquid fuel – would be a valuable process environmentally and for the fuel industry. In nature, bacterial enzymes particulate and soluble methane monooxygenases (MMOs) can effectively and selectively catalyze the direct oxidation of methane to methanol using oxygen at copper and iron active sites, respectively. Unfortunately, the bacteria that make these enzymes are difficult to cultivate, so a synthetic mimic would be a useful alternative. Synthetic catalysts, however, have great difficulty in selective oxidation of unactivated C–H bonds. Our research intends to fabricate tricopper and diiron complexes that can effectively mimic MMO catalysis. We hypothesize that by inserting small metal catalysts into a polymeric scaffold, our metallo-polymer hybrid systems will simulate the active site environment of the enzyme and facilitate comparable reactivity. We have successfully reproduced syntheses for two small organic molecules that will bind directly to the metals
(N,N’-bis-(2-pyridylmethyl)-(2-hydroxy-3-carbonyl-5-methylbenzyl)-1,3-propanediamine-2-ol) (1) and N,N-dimethyl-N’(pyrid-2-ylmethyl)-ethylenediamine (2). Both synthetic routes involved an imine condensation followed by a reduction of the imine to an amine. The synthesis of (2) was completed using these steps. (1) required a subsequent substitution reaction between 2-chloromethyl-4-methyl-6-formylphenol and the relevant amine product. Modification of the literature procedures focused on changing purification techniques, including extraction and column chromatography, which resulted in a roughly 40% increase in isolated yield. Nuclear magnetic resonance spectroscopy and mass spectrometry were used to analyze the reaction products and confirmed the successful synthesis of (1) and (2). Addition of an alkene group to a starting material for (1) has been achieved via Stille coupling reaction. Our next step is to synthesize and covalently connect our alkene-modified metal complexes to synthetic polymers which will serve as scaffolds for the desired reactivity, aiming to enhance both the catalytic activity and selectivity. We will test the small molecules (metallated versions of (1) and (2)) as benchmarks for our metallo-polymer hybrid systems by measuring their relative efficiency and selectivity in alkane oxidation reactions. We acknowledge San Jose State, CSUPERB, and the NIH RISE program for funding.
Poster #: 288
Campus: CSU Long Beach
Poster Category: Synthetic Chemistry
Keywords: organophosphates, chiral shift reagent, diastereotopic methyl groups
Project Title: A Rapid Determination of Enantiopurity of P-Chiral Compounds With Eu(hfc)3: the Terminal Methyl Group Signal is Most Diastereotopically Affected
Author List:
Ly, Phillippe; Graduate, chemistry and biochemistry, California State University, Long Beach, Presenting Author
Tran, Trina; Graduate, chemistry and biochemistry, California State University, Long Beach
Pace, Codi; Undergraduate, chemistry and biochemistry, California State University, Long Beach
Nakayama, Kensaku; Faculty, chemistry and biochemistry, California State University, Long Beach
Abstract: Compounds with chiral phosphorus atom centers are playing increasingly important roles in the development of pharmaceutical drug candidates, agrochemicals, and chiral ligands in catalysis. In our search for specific cholinesterase inhibitors for the treatment of Alzheimer’s Disease, we have been investigating a chiral organophosphorus scaffold—the alkyl 2-(dimethylamino)ethyl phenyl phosphate. A variety of methods are currently available to determine the enantiomeric excess (ee) of chiral organophosphorus compounds, including chiral high-performance liquid chromatography and vibrational circular dichroism; however, these are often time-consuming methods due to condition optimization and also limited due to instrumentation availability. Herein, we describe the use of proton nuclear magnetic resonance in the presence of the lanthanide shift reagent, Eu(hfc)3 (Europium tris[3-(heptafluoropropylhydroxymethylene)-d-camphorate]) to assay the ee’s for these phosphate triesters. Previous studies employing this reagent focused on the alpha-carbon protons of the phosphate triesters to display diastereotopic behavior. However, in the fore-mentioned phosphate triesters, the terminal methyl group of the alkyl substituent was observed to have the most highly chemically shift-affected signal and proved to be most useful in the determination of ee. We hypothesize that this contrasting behavior from earlier reports is due to the Lewis-basic nitrogen atom coordinating to the Lewis-acidic europium atom center. To further probe this effect, we have synthesized a series of racemic chiral phosphate triesters with various alkyl chain lengths and heteroatom-containing substituents. Our results show a trend where more Eu(hfc)3 is needed to determine the ee for analogs of the alkyl 2-(dimethylamino)ethyl phenyl scaffold with longer alkyl chain lengths: ethyl (6 mg), butyl (10 mg), and hexyl (25 mg). In addition, the 2-(trimethylammonium)ethyl series required much less shift reagent to determine ee (6 mg for both butyl and hexyl) than the 2-(dimethylamino)ethyl analogs, indicating that charge may also play a large role in affecting how each enantiomer are magnetically influenced in the ligand-Lewis acid complex. In summary, this work describes a convenient method to rapidly determine the ee’s of P-chiral compounds using Eu(hfc)3, in which protons most affected are those most distal to the phosphate moiety.
Poster #: 289
Campus: CSU Fresno
Poster Category: Synthetic Chemistry
Keywords: Gold catalysis, benzothiophenes, organic synthesis
Project Title: Scope of the Gold(I)-IPr-OH-Catalyzed Synthesis of Benzo[b]thiophenes
Author List:
Keophimphone, Bagieng; Undergraduate, Chemistry and Biochemistry, California State University, Fresno, Presenting Author
Sanchez, Melissa; Undergraduate, Chemistry and Biochemistry, California State University, Fresno, Presenting Author
Muchalski, Hubert; Faculty, Chemistry and Biochemistry, California State University, Fresno
Abstract: Benzothiophenes and their derivatives are important molecular scaffolds that can be synthesized synthetically or naturally. The applicability of these molecules serves a biological importance within the developing health field. Benzothiophene-based compounds in pharmaceuticals have been used to treat and prevent diseases such as osteoporosis, asthma attacks, and athlete’s foot. A variety of methods have been published to date but they often require costly reagents or harsh reaction conditions. We discovered the synthesis of benzothiophenes from 2-alkynyl thioanisoles which uses Au-IPr-OH (N, N′-Bis(2,6-diisopropylphenyl)imidazol-2-ylidene gold(I) hydroxide) as a catalyst. The gold(I) complex is beneficial to the synthesis as it is stable to the moisture in the air and is commercially available. Here we report the results of evaluation of the substrate scope for this new reaction. First we synthesized a suite of disubstituted 2-alkynyl thioanisoles containing electron-rich and electron-poor substituents (both alkyl and aryl) as well as bulky groups. Then we tested each compound using optimized reaction conditions using only 1 mol% of gold(I)-IPr hydroxide in toluene at 100 °C. Consumption of the starting material was measured using quantitative NMR spectroscopy using methyl-2,5-dinitrobenzoate as internal standard. The reaction tolerates a broad range of substrates and can be carried out in air using commercial grade reagents and solvents. This project was funded by Fresno State ASI and Presidents’ Commission Scholars Program from CSUPERB.