Search Poster #: 1 Campus: California State University, Long Beach Poster Category: Agriculture/Biofuels/Environment Keywords: isotopes, nitrogen, nitrous oxide Project Title: Assessing Nitrous Oxide Cycling in the Coastal Waters of the Eastern Tropical South Pacific Using Stable Isotopes Author List: Chen, Jacquelin; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Gluschankoff, Noah; Graduate, Earth System Science, Stanford University
Casciotti, Karen; Faculty, Earth System Science, Stanford University
Abstract  Nitrous oxide (N2O) is a climatically relevant greenhouse gas that can be produced in the ocean and emitted to the atmosphere, where it can later lead to ozone depletion. Previous studies show high accumulations of N2O above the oxygen minimum zone (OMZ) of the Eastern Tropical South Pacific (ETSP), but the relative roles of the microbial pathways that dictate N2O cycling vary regionally. The purpose of this study is to identify the primary pathways controlling N2O cycling in the coastal waters of the ETSP. In this study, N2O concentrations and its isotopes, as well as O2 concentrations and the isotopic compositions of nitrate and nitrite, were analyzed at three stations between the shallow coastal shelf and the shelf slope along the GEOTRACES GP16 Zonal Transect. Our results demonstrated a rapid decline in O2 concentrations coinciding with accumulation of N2O. This N2O accumulation is estimated to support significant fluxes of N2O to the atmosphere at our stations. N2O isotopocule results illustrate intense N2O cycling, driven by a combination of incomplete denitrification and hybrid N2O production in the oxycline. In the anoxic waters of the ODZ N2O isotopes indicate concurrent N2O production and consumption through changes in 𝛿15N⍺, 𝛿18O, and 𝛿15Nβ. Overall, N2O cycling in the coastal ETSP waters appears to be mainly driven by denitrification, with a smaller role from hybrid formation. | Poster #: 2 Campus: California State University San Marcos Poster Category: Agriculture/Biofuels/Environment Keywords: Cellulase, Biofuels, Bioprospecting Project Title: Novel Carbohydrate-active Enzymes from the Cow Rumen Microbiome as Tools for Biofuel Production Author List: Ponce, Melanie; Graduate, Biological Sciences, California State University San Marcos, Presenting Author
Escobar, Matthew; Faculty, Biological Sciences, California State University San Marcos Abstract The global impacts of climate change have highlighted the importance of carbon neutral energy sources such as biofuels. Cellulose is one of the most abundant biopolymers on the planet, and cellulose-rich agricultural and forestry waste products are very promising resources for biofuel production. However, a primary limitation in the commercial production of cellulosic biofuels is the inefficiency and high cost of degrading cellulose-rich plant biomass into glucose. Thus, the focus of this study was to characterize novel carbohydrate-active enzymes (CAZYmes) that can efficiently break down plant cell wall carbohydrates for the purpose of biofuel production. The cow rumen is a rich reservoir of cellulolytic microorganisms that produce novel, uncharacterized CAZYmes. Using a previously published cow rumen metagenome dataset, we identified 2,232 sequences containing glycoside hydrolase 5 (GH5), GH8, GH9, or GH 48 protein domains, which are characteristic of cellulase enzymes. We then used NCBI Batch CD Search to characterize the domain structure of these sequences. 125 genes contained multiple carbohydrate-relevant protein domains, suggesting that they may encode multi-functional cellulases, which have been shown to be highly efficient in cellulose degradation. PCR primers were designed to amplify 36 of these multi-domain sequences, and we were able to successfully amplify 11 novel CAZYme sequences from cow rumen DNA. We are currently cloning these sequences into the pET100-TOPO vector for use in recombinant protein production in Escherichia coli. We will characterize the enzyme kinetics and substrate specificity of these putative multi-functional cellulase enzymes, and compare their performance to commercially available cellulase enzyme cocktails that are currently used for biofuel production. | Poster #: 3 Campus: California State University, Fullerton Poster Category: Agriculture/Biofuels/Environment Keywords: Aquaculture, Chlamydomonas reinhardtii, Classical Strain Improvement Project Title: Algae for Aquaculture: Increased Protein and Lipid Production in Chlamydomonas reinhardtii Using Classical Strain Improvement Techniques Author List: Salman, Tanya; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Xia, Angela; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Cohen, Amybeth; Faculty, Biological Science, California State University, Fullerton
Abstract Over the past twenty years, global farmed-fish production has increased by more than 500%, necessitating a parallel increase in fish food production. Historically, fish meal and fish oil (FMFO) supplied farm-raised fish with the needed essential proteins and omega-3 fatty acids. However, using ocean resources for FMFO is unsustainable. This project aims to develop high-fat and high-protein strains of the single-celled, green microalga Chlamydomonas reinhardtii through non-GMO practices, which could be used to supplement FMFO. We employed the classical strain improvement (CSI) techniques of UV mutagenesis with nitrogen-deficient induced gametogenesis and mating, as they had been successfully used to produce strains of C. reinhardtii with an increased chloroplast-protein content (Fields et al., 2019) and survivability in high salinity conditions (Takouridis et al., 2012). In our study, five pairs of wild-type (WT) C. reinhardtii strains were subjected to CSI techniques, establishing fifteen genetically diverse offspring populations: five from sexual recombination during mating and 10 from UV mutagenesis. Ten isolated colonies from each population were surveyed for crude protein and lipid content, using a crude protein extraction and Bradford assay and BODIPY 493/503 lipid staining and fluorescence assay, respectively, to isolate the first set of top-producers. The five selected strains from mating underwent UV mutagenesis, while the 10 strains that were first exposed to UV light underwent mating in order to develop the second round of genetically recombinant populations. Crude protein and lipid assays of the newly isolated colonies revealed eight high protein and 18 high lipid strains. The two top-producing strains have either a 57.6% increase in total protein or a 150.2% increase in total lipids compared to the WT parents. The next steps will involve fluorescent-activated cell sorting of desired populations to screen for strains with an even more significant increase in lipid production than the WT parents, followed by confocal microscopy of BODIPY 493/503 lipid-stained cells, SDS-PAGE of extracted proteins, and determination of the amino acid and lipid profiles in the selected strains.
This project is supported by a CSUPERB Faculty-Graduate Student Collaborative Research Grant, the COAST Undergraduate Student Research Support Program, and funding from the Department of Biological Science at CSU Fullerton.
| Poster #: 4 Campus: California State University, Northridge Poster Category: Agriculture/Biofuels/Environment Keywords: Arsenic, Halobacterium , Bioremediation Project Title: Cloning and characterization of arsenite efflux transporter metallochaperone arsD- related protein from Halobacterium salinarum Author List: Patel, Lipi; Graduate, Biology, California State University, Northridge, Presenting Author
LNU, Shaurya; California State University, Northridge, Presenting Author
Perl, Scott; NASA JPL
Basu, Chhandak; Faculty, Biology, California State University, Northridge Abstract Halobacterium salinarum is a model organism to study microbial stress physiology because of its ability to survive in extremes of environmental conditions, including high salinity, UV light, and heavy metals like arsenic. Arsenic is one of the most toxic metals known to have devastating effects on humans, such as cancer, low mortality rate, and inhibit plant growth and photosynthesis. It also is a significant contaminant in many food commodities such as seafood, poultry, and rice. Halobacterium salinarum comprises genes responsible for heavy metal resistance and enzymes for detoxifying heavy metals. Therefore, this trait of Halobacterium could be used for promising bioremediation of environments contaminated with highly toxic metals due to urbanization and industrialization. An effective way of performing bioremediation is phytoremediation, which treats pollutants or wastes by using plants to remove, degrade, or stabilize unwanted substances such as toxic metals by sequestering them from the soil. It is very important to remediation practitioners because they can put plants to work and save money on excavation costs. The soil structure remains preserved as well. This method could be used by people in developing countries where comparatively, such problems are more prevalent due to a lack of resources. The project aims to clone and characterize the arsenite efflux transporter metallochaperone arsD- related protein (GenBank accession number NC_001869.1) from halobacterium and express it in hyperaccumulator plants like tobacco. Once this gene is expressed, these plants are expected to sequester the arsenic compounds and ameliorate soil health. We have successfully established the required conditions for the growth of Halobacterium salinarum and transformed it using pBI121 by the osmotic shock method. We are currently in the process of cloning the arsenite efflux transporter gene from Halobacterium and inserting it into the pBI121 plasmid vector by replacing the GUS reporter gene. The recombinant plasmid will be transferred to Arabidopsis thaliana by the ‘floral dip’ method. Arabidopsis plants will be used as a ‘proof of concept’ to evaluate the plants’ abilities to remove arsenic from soil. The project’s long-term goal is to evaluate a transgenic tobacco plant expressing the arsenite efflux transporter gene for its ability to remove arsenic from soil. | Poster #: 5 Campus: California State University, Northridge Poster Category: Agriculture/Biofuels/Environment Keywords: Algae, Dunaliella salina, Biofuel Project Title: Production of cold-tolerant strains of Dunaliella salina through genetic transformation with Agrobacterium tumefaciens Author List: Hein, MacKenzie; Graduate, Biology, California State University, Northridge, Presenting Author
BASU, CHHANDAK; Faculty, Biology, California State University, Northridge Abstract Genetically modified microalgae have considerable potential as bio-factories for the production of value-added compounds such as biofuels, glycerol, and carotenoids. However, production may be disrupted by the sensitivity of microalgae to conditions that inhibit their growth, such as high salinity and low temperatures. Many microalgae also possess a cell wall, which must be removed before the cells can be transformed. Dunaliella salina is a eukaryotic microalgae species with several traits that make it attractive as a platform for genetic modification. This microalga is also halotolerant,i.e., able to survive across a wide range of salinities. This means that the salinity of water used for the aquaculture of this algae does not need to be precisely maintained, and the cells can be grown in seawater or hypersaline brine. However, observations in our lab indicate that this algae’s growth can be severely inhibited by low temperatures, meaning that cultivation in cold or temperate climates may prove difficult. A possible solution may involve the genetic transformation of Dunaliella salina with genes from psychrophilic microalgae species, which confer the ability to grow at low temperatures. We have already succeeded in the cultivation of Dunaliella salina in the lab, and we have compared the growth of the organism at both room temperature and 4oC. We have used electroporation to transform Agrobacterium tumefaciens with PBI121 plasmids containing a GUS reporter gene to test the efficacy of the transformation protocol. We are currently in the process of genetically transforming the algae via co-cultivation with Agrobacterium tumefaciens harboring a potentially cold-tolerant ferredoxin gene from Chlamydomonas sp. UWO241. If the transformation is successful, it will allow us to study the potential benefits cells resistant to multiple types of extreme conditions may have for biomanufacturing. Microalgae grown for the production of valuable compounds are cultivated in open-air pools. Engineering microalgae to grow at low temperatures would allow them to be grown continuously year-round in temperate climates, without interruption by decreases in temperature during fall and winter. | Poster #: 6 Campus: California State University, Monterey Bay Poster Category: Agriculture/Biofuels/Environment Keywords: arbuscular mycorrhizal fungi, agriculture, amf Project Title: Evaluating the impacts of varying healthy soil practices on arbuscular mycorrhizal fungi spore presence Author List: Martinez, Katherina; Undergraduate, Biology and Chemistry, California State University, Monterey Bay, Presenting Author
Hynes, Meagan; Faculty, Biology and Chemistry, California State University, Monterey Bay
Haffa, Arlene; Faculty, Biology and Chemistry, California State University, Monterey Bay
Kortman, Stefanie; Staff, Science & Environmental Policy, California State University, Monterey Bay Abstract Arbuscular mycorrhizal fungi (AMF) are soil microorganisms that can form mutualistic symbiosis with plants. The AMF acts as an extension of the plants’ root system to aid in nutrient exchange and increased resilience against stressors. Utilization of commercial AMF inoculum in soil health practices may prove beneficial to combat climate change effects, sustain agricultural production, and maintain soil aggregate stability. This research aims to see if applications of mycorrhizal inoculum to the soil would yield an increased presence of AMF spores in a commercial organic cropping system of romaine lettuce and celery. Treatments of AMF inoculum, AMF inoculum plus compost, as well as a control were used in determining changes to the overall AMF spore count in the soil. From 2020 to 2022, soil core samples were collected, extracted, and are currently being counted for spore presence. Currently, there are no significant findings regarding the different treatments. However, at this time, an overall decrease in spore counts was found from preplant to harvest for the fall core samples in 2020 and 2021 suggesting utilization of mycorrhizal spores by the plants over the growing season. AMF inoculation was confirmed for Fall 2020, 2021, and Spring 2021 by analyzing root samples from plants in each treatment. The spring samples need to be completely evaluated before we can reach a definite conclusion regarding the findings on the different treatments and sampling time. | Poster #: 7 Campus: California State University, Northridge Poster Category: Agriculture/Biofuels/Environment Keywords: climate change, Permafrost Project Title: Isolation of Polarmonas spp. from ancient thawing permafrost Author List: Sarkisyan, Eleonora; Undergraduate, Biology, California State University, Northridge, Presenting Author
Mackelprang, Rachel; Faculty, Biology, California State University, Northridge Abstract Permafrost is soil that remains permanently frozen throughout the year and is located in polar regions of the Earth. Climate change is causing permafrost to thaw on a large scale. Permafrost contains large amounts of organic carbon, and microbes are able to convert the carbon into greenhouse gasses when it is thawed. In this study permafrost samples, frozen for 5,000 and 40,000 years, were collected from central Alaska and transplanted into the active layer (soil overlying permafrost that freezes and thaws annually) in order to stimulate thaw. After the permafrost samples were collected and transported to our laboratory. Initial studies from our samples show that Polarmonas spp. (phylum Proteobacteria, family Comamonadaceae) are highly abundant in thawed permafrost. Previously cultivated members of this genus are known to be psychrotolerant, which means they are capable of growth during low temperatures.
To better understand the physiology of these potentially important bacteria, we are attempting to culture Polarmonas spp. from thawed permafrost in the laboratory. After the species are cultured and the genome sequenced, we will better understand survivability and growth characteristics.These data will yield insight into the activity and carbon degradation processes in a species that may be crucial in producing greenhouse gasses during permafrost thaw.
| Poster #: 8 Campus: California State University San Marcos Poster Category: Agriculture/Biofuels/Environment Keywords: marine science, plankton, microscopy Project Title: Investigating the relationship between nutrient levels and autotrophic and heterotrophic plankton biomass off the coast of southern California Author List: Gutierrez, Luis A.; Undergraduate, Biology, California State University San Marcos, Presenting Author
Garcia, Anissa M.; Graduate, Biology, California State University San Marcos, Presenting Author
Taniguchi, Darcy A.A.; Faculty, Biology, California State University San Marcos
Abstract Marine drifters made of one cell, known as single-celled plankton, are the base of the oceanic food web and play a major role in the movement of elements and compounds around the ocean. These single-celled plankton can obtain their energy through photosynthesis, known as autotrophs, or through consumption, known as heterotrophs. Autotrophic plankton rely on nutrients such as phosphate, nitrogen, and silicate for growth and cell maintenance. In turn, heterotrophic plankton consume autotrophic and other heterotrophic plankton. Since nutrient concentrations can have a direct impact on planktonic abundances, we aimed to study the relationship between these two factors in a gradient from near the coast to the open ocean. Samples of plankton and nutrient concentrations were taken off the coast of southern California during 2020 and 2021 in locations ranging from nearshore to offshore. Two different forms of microscopy, brightfield and epifluorescence, were used to image, identify, and categorize cells into two groups, diatoms (autotrophic plankton) and non-diatoms (a mixture of autotrophic and heterotrophic plankton). We used the image analysis software ImageJ to obtain cell size measurements, which we then used to calculate the total carbon biomass of both groups. Our results indicated that as nutrient levels increased, so did the total carbon biomass of diatoms. Additionally, results showed no correlation between the total carbon biomass of non-diatoms and nutrient levels. Future analysis of additional microscopy samples will allow us to see the strength of these relationships over a greater amount of time and space. | Poster #: 9 Campus: California State University, Long Beach Poster Category: Agriculture/Biofuels/Environment Keywords: Geometric Morphometrics, Justicia, Reproductive Biology Project Title: Morphological Variation in Justicia (Acanthaceae) Corolla and Pollen Author List: Wang, Yuting; Graduate, Biological Sciences, California State University, Long Beach, Presenting Author
Fisher, Amanda; Faculty, Biological Sciences, California State University, Long Beach Abstract Justicia (Acanthaceae) species have a wide variety of flower forms and have adapted to many types of pollinators in the western hemisphere including bats, bees, butterflies, hummingbirds, and moths. Justicia species pollinated by the same animals tend to share similar corolla shapes and colors. Before molecular science, botanists used to classify species based on morphology. For instance, some moth-pollinated Justicia were formerly classified in the genus Siphonoglossa based on their long and narrow corolla tubes. However, a molecular phylogeny of Justicia found that Siphonoglossa is not monophyletic and there have been many transitions between pollinator types among closely related Justicia species. This means that similarities in Justicia corolla morphology evolved through convergence. Other flower characteristics such as pollen size and shape may have also co-evolved with animal pollinators. My objectives are to compare: (1) corolla shape and size and (2) pollen shape and size among Justicia species in the ‘sessile-axillary inflorescence’ and the Dianthera/Sarotheca/Plagiacanthus clade. The species in these clades are known to have bee, hummingbird, and lepidopteran pollination. One-way ANOVA and Tukey’s tests detected variation in corolla and pollen length, width, and length-to-width ratio among Justicia species. Supporting my prediction, lepidopteran pollinated Justicia species have a significantly higher corolla length-to-width ratio when comparing to Justicia relatives that are pollinated by bee, hummingbird, or insect generalists. Results from geometric morphometric analyses show differences in corolla shape of Justicia species with different pollinators. Lepidopteran pollinated Justicia species have a narrower corolla tube and a higher corolla tube opening while generalist pollinated species has a larger corolla pouch. I will compare pollen shape in Justicia species pollinated by different animals using outline analyses. I expect that lepidopteran-pollinated Justicia species will have pollen that is more similar in size, shape, and texture compared to their close Justicia relatives that are bird or bee pollinated. This study will help to advance our knowledge of flower-animal co-evolution and transitions between different animal pollinators in western hemisphere Justicia. This project is funded by National Science Foundation Award #1754792 and a CSUPERB Faculty-Student Collaboration Grant. | Poster #: 10 Campus: California State University, San Bernardino Poster Category: Astrobiology Keywords: Hot spring, Thermophile, Exobiology Project Title: Growth of Caldatribacterium (OP9) in pure culture and in co-culture with sulfate-reducing bacteria Author List: Torres, Aries; Graduate, Biology, California State University, San Bernardino, Presenting Author
Alvarado, Toshio; Graduate, Biology, California State University, San Bernardino
Dodsworth, Jeremy; Faculty, Biology, California State University, San Bernardino
Abstract The Atribacterota (formerly called Atribacteria or OP9) is a phylum of bacteria whose members are found in many anaerobic environments such as hot springs, non-thermal ocean sediments, and petroleum reservoirs. Currently there is only one cultivated representative in this entire phylum, and little is known about the physiology and ecology of these microbes. Members of our lab recently isolated another member of this phylum, Caldatribacterium, from Great Boiling Spring, NV. During initial enrichment, Caldatribacterium appeared to be growing in co-culture with a member of the genus Thermodesulfobacterium, a sulfate-reducing thermophile, in media with fucose as a sole carbon source. Caldatribacterium was eventually isolated by including yeast extract in addition to fucose, but the isolate could not grow on fucose alone. This led to the hypothesis that the sulfate-reducing bacterium may be supporting growth of Caldatribacterium. To test this, co-cultures were made of Caldatribacterium with three different species of Thermodesulfobacterium and a species of Thermodesulfovibrio, which is also a thermophilic sulfate reducer. All four of these sulfate reducing bacteria were able to support growth of Caldatribacterium (2.1-6.8*10^7 cells/mL) in co-culture on fucose alone, whereas none of these microbes can grow on their own under these conditions (<10^6 cells/mL). This raised the question of what benefit the sulfate-reducer might provide to Caldatribacterium. The vitamin solution that is routinely used for the Caldatribacterium and co-cultures is typically filter-sterilized, but we noticed that some of the vitamins may not have been fully soluble and may have been retained on the filter, not allowing for growth. To test this, the vitamin solution was autoclaved rather than filter sterilized. The autoclaved vitamin solution allowed for growth of Caldatribacterium on fucose alone (0.3-1.2*10^8 cells/mL) to similar levels obtained when yeast extract is included (0.3-2.9*10^8 cells/mL), indicating that the autoclaved vitamins are providing or transforming a compound required by Caldatribacterium in the vitamin solution. These data suggest that the partner organism might be producing a vitamin needed by Caldatribacterium to grow in co-culture. Learning more about the nutritional requirements of Caldatribacterium may help inform cultivation of other members of the Atribacterota present in different environments.
Supported by NASA Exobiology grant 80NNSC17KO548.
| Poster #: 11 Campus: California State University, San Bernardino Poster Category: Astrobiology Keywords: Archaea, Astrobiology, Thermophile Project Title: Laboratory Cultivation of and Substrate Utilization by the Novel Thermophilic Lineage Gearchaeales Author List: Saldivar, Walter; Graduate, Biology, California State University, San Bernardino, Presenting Author
Moya, Jennifer; Undergraduate, Biology, California State University, San Bernardino
Dodsworth, Jeremy; Faculty, Biology, California State University, San Bernardino
Abstract The majority of microbes on Earth have yet to be grown in pure culture and studied in the laboratory. Cultivation-independent genomic methods like metagenomics can predict what these microbes can do, but cultivation is still valuable in assessing these predictions. Uncultivated microbes are truly diverse, especially within hot springs. Great Boiling Spring (GBS) in Nevada is home to many uncultivated microbes, including the archaeal order Gearchaeales, which is found in other hot springs around the world. Previous students established laboratory cultures from GBS that contained Gearchaeales at ~2% abundance based on 16S rRNA gene tags, along with other novel microbes. These cultures have 0.02% casamino acids as a carbon source, ~10% oxygen, are incubated at 80 ℃, and are transferred every 3 weeks. This mixed culture has been maintained since 2016 and we were unsure if Gearchaeales was still present in the culture. The goals for this project were to confirm the persistence of Gearchaeales in the cultures and to find conditions that will further enrich for Gearchaeales. Quantitative polymerase chain reaction (qPCR) was used to determine Gearchaeales abundance on DNA extracted from cultures grown under different conditions in triplicate, including incubation temperature and time, oxygen levels, and growth substrates. The qPCR confirmed that Gearchaeales was present at ~1.2-2.8*10^7 16S rRNA gene copies/mL culture and that 3-4 weeks of incubation was required to maintain stable populations. The abundance of Gearchaeales increased as culture temperature was increased from 75-85 °C, but were not detected at 90 °C. Tests with different oxygen concentrations confirmed that 10% oxygen yielded the best growth (1.2-4.0*10^7 copies/mL), with 20% oxygen (fully aerobic) showing reduced abundance (6.5-9.9*10^6 copies/mL); microaerobic (2.5-5%) or anaerobic conditions did not support growth (<10^5 gene copies/mL). Starch (5.6-7.4*10^6 copies/mL) and a mixture of butyrate, propionate, and acetate (BPA; 0.6-3.6*10^6 copies/mL) as alternative carbon and energy sources supported growth, but at lower levels compared to casamino acids (1.5-3.7*10^7 copies/mL), although overall cell abundance was lower with BPA, suggesting enrichment. Further experiments will combine some of these conditions to help further enrich for Gearchaeales, which will help us to better understand this lineage and possibly lead to a pure culture.
Supported by NASA Exobiology grant 80NNSC17KO548.
| Poster #: 12 Campus: San Francisco State University Poster Category: Astrobiology Keywords: hot springs, microdiversity, metagenomics Project Title: Metagenomics-Based Analysis of Population-Level Genomic Variation in Hot Spring Microbial Communities Author List: Hogan, Daniel R.; Graduate, Biology, San Francisco State University, Presenting Author
Williams, Andre's W.; Graduate, Biology, San Francisco State University, Presenting Author
Salcedo, Rebecca S. R.; Stanford University
de la Torre, José R.; Faculty, Biology, San Francisco State University Abstract Metagenomics, sequencing genomic DNA from entire microbial communities, has altered our understanding of the microbial world, revealing unsuspected levels of biodiversity. Most metagenomic analyses have focused on obtaining metagenome-assembled genomes, which represent the consensus genome sequence of organisms in the community. However, these datasets also contain information about the genetic variation present within each species in the community. To date, the extent of this microdiversity, along with the environmental and evolutionary forces driving it, have largely been ignored. Prior work in our lab examined the microdiversity of natural populations of thermophilic ammonia-oxidizing archaea (ThAOA) using metagenomic datasets sequenced with Illumina short-read technologies (reads of 100-150 bp). We found great variability in the amount of genetic diversity of ThAOA populations in hot springs across three continents, as indicated by the density of single nucleotide polymorphisms (SNPs). Some populations (Spain) appeared almost clonal, with little or no genetic variation across the entire genome. In contrast, we found that populations in the United States (Nevada) and China (Yunnan) showed significant diversity across all loci. We have now examined whether other dominant microorganisms in the samples show similar patterns of diversity. In the Spanish community, the second most abundant organism was a close relative of Thermoflexus hugenholtzii. Like the ThAOA in this sample, we found remarkably low numbers of SNPs for T. hugenholtzii, suggesting that the microbial community in this engineered hydrothermal system may be extremely new. In springs with genetically diverse populations of ThAOA, we likewise found significant SNP densities across the genomes of other abundant microorganisms. More recently, we have looked at genomic variability at larger scales within populations. Flexible genomic islands, often containing multiple genes or operons, result from the acquisition by individual cells of large genomic fragments by horizontal gene transfer. Genes in flexible islands have higher proportions of non-synonymous to synonymous changes when compared to genes in the core genome. These results suggest different selective pressures may be acting on genes found in the core or flexible regions of the genome. In the future, we will explore the use of long-read sequencing technologies to improve our assessments of genomic variability in these communities. | Poster #: 13 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: inflammation, blood plasma ceruloplasmin, rates of turnover Project Title: The role of the copper-containing blood plasma protein, ceruloplasmin, in inflammation Author List: Savage, Cannon; Graduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author, Eden Award Nominee
Sanqui, Cossette ; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Perez, Madelynn; Fullerton College
Linder, Maria C; Faculty, Chemistry and Biochemistry, California State University, Fullerton Abstract Ceruloplasmin (Cp) is a 132 kDa Cu binding glycoprotein in blood plasma involved in numerous processes including delivery of copper to cells and neutralization of reactive oxygen species. It is largely produced by liver hepatocytes, with initial synthesized as copper-less “apo” Cp in the rough endoplasmic reticulum, then glycosylated, with Cu added in the trans-Golgi network before secreted into the circulation as “holo” Cp. Upon donating its Cu to cells, Cp reverts to apoCp and is removed from the circulation by the liver upon desialylation and endocytosis. In addition to its Cu transport duties, there is a clear role for Cp in the acute phase response of inflammation. We have been studying the time-course of the Cp response to inflammation using the turpentine inject model in rats, following changes in Cp enzyme activity, mRNA, and proportions of holo to apo Cp. After a single turpentine injection, Cp mRNA levels increased 3.5 fold, peaking at 16 h and returning to normal after 24 h. a similar pattern was observed for kidney Cp mRNA. This spike in expression was followed by a tripling of Cp enzyme activity in the blood, which reaches a maximum by 48 h, implying an increase in transcription and translation. Interestingly, Cp activity in the blood remained elevated for upwards of 7 days, and not returning to control levels until 14 days. While the normal half-life of Cp is not clearly established, but some evidence suggesting ~10 h, our findings imply that the inflammatory response while transiently increasing the rate of Cp synthesis reduces the rate at which Cp is removed from the blood. Our next objective is to test this idea by measuring the rates of synthesis and degradation of Cp in control and turpentine-treated rats using incorporation and retention of 14C-leucine and 67Cu in circulating Cp over time, and for which we have developed a new Cp isolation procedure. If correct, this will add a whole new dimension to our knowledge of Cu metabolism and immunity. Samples from the time-course study described were also analyzed for effects of inflammation on the ratio of holo to apo Cp, after developing a new and reproducible native PAGE immunoblotting procedure. Ratios of holo to apo were markedly enhanced, which is consistent with the idea that the active Cu-dependent form of Cp is required in inflammation.
| Poster #: 14 Campus: California State University, Stanislaus Poster Category: Biochemistry Keywords: acetanilide, saturation mutagenesis, toluene o-xylene monooxygenase Project Title: Tuning the specificity of toluene o-xylene monooxygenase for acetanilide metabolism Author List: Frazer, Eli; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author, Nagel Award Nominee
Vardar Schara, Gönül; Faculty, Chemistry, California State University, Stanislaus Abstract The biocatalyst toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1 belongs to the great family of bacterial multicomponent monooxygenases and has been shown to have a potential for biotechnological and environmental applications. Acetanilide, which was once a commonly used drug, is mostly metabolized into 4-acetamidophenol (4-AAP) in the body, which gives its analgesic and antipyretic properties. 4-AAP, also known as acetaminophen and sold under the name Tylenol, has replaced acetanilide in therapy and its derivative 2-AAP has been shown to have therapeutic potential. In this study, our goals were to examine the metabolism of acetanilide with native ToMO and generate ToMO variants with fine-tuned specificities using protein engineering. Active site residues were subjected to combinatorial saturation mutagenesis approach of protein engineering for the very first time to achieve a comprehensive study by exploring all the substitutions for these codons with the goal of seeking further improvements in ToMO specificity. Oxidation regiospecificities were determined using reverse-phase high-performance liquid chromatography on a C18 column using a water/methanol/2-propanol solvent system. We found that ToMO successfully metabolized acetanilide and produced mixtures of 2-AAP, 3-AAP, and 4-AAP. Total of four ToMO variants were isolated from the saturation library using a powerful agar plate screening method. We found that the selectivity of 2-AAP, 3-AAP, and 4-AAP was enhanced by up to 3.7-, 1.5-, and 2.5-fold by ToMO variants relative to the native enzyme. This study confirms the advantages of the semi-rational approach for protein engineering, shows the potential of non-human ToMO and its variants in drug metabolism applications, and adds to the list of research on probing this remarkable enzyme. Furthermore, the results of the present study show the capacity of native and engineered ToMOs for regiospecific oxidation of acetanilide for the synthesis of acetaminophen and its derivatives using environmentally-benign biological methods rather than currently used traditional chemical processes. This project is supported by the National Institutes of Health Support of Competitive Research Program. | Poster #: 15 Campus: California State University, Northridge Poster Category: Biochemistry Keywords: Momordica Charantia, gluconeogenesis, HepG2 cells Project Title: The Effect of Bitter Melon (Momordica Charantia) Extract on the Regulation of Gluconeogenesis in Liver Cells Author List: Thomas, Christi; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author, Nagel Award Nominee
Medh, Jheem; Faculty, Chemistry and Biochemistry, California State University, Northridge Abstract Background: Diabetes is widely prevalent disease. In 2022, the Center for Disease Control and Prevention reported that 11.3% of the US population (about 28.7 million people) were diagnosed with diabetes and 96 million adults had prediabetes. In Asia and Africa, the bitter melon fruit is traditionally used as a medicinal supplement for managing type 2 diabetes.
Objective: The purpose of this study was to examine the effects of bitter melon extract on the transcription of regulatory enzymes of gluconeogenesis in a human liver cell line (HepG2 cells). The phenolic content and antioxidant capacity of the bitter melon extract will also be determined.
Methods: The juice of bitter melon was squeezed from both the skin and the flesh of the fruit.
The phenolic content was measured by the Folin-Ciocalteu reagent (FCR) Assay, which is a colorimetric assay using Gallic Acid as a standard. The Ferric Reducing Antioxidant Power Assay (FRAP) Assay, also a colorimetric assay, was used for detecting antioxidants at multiple capacities by the reduction of iron (III) to iron (II).
HepG2 cells were incubated without or with the bitter melon extract for 2-4 days. Total RNA was isolated and specific primer pairs were used to amplify phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6PC), the regulatory enzymes of gluconeogenesis. Both endpoint reverse transcription polymerase chain reaction (RT-PCR) and quantitative polymerase chain reaction (qPCR) were used for the specific amplification, detection, and quantification of PEPCK and G6PC. beta-actin was amplified as a control house-keeping gene.
Results: The phenolic content of both bitter melon skin and flesh was similar at about 1 mg/mL.
The FCR and FRAP assays indicated that bitter melon had high levels of phenolic content (750 to 1500 mg/L) and antioxidant capacity (20-30 g/mL). Endpoint PCR qualitatively showed that the transcription of PEPCK and G6PC were reduced in HepG2 cells treated with bitter melon extract, compared to controls. Analysis of Ct values obtained by qPCR by the delta-delta-Ct method showed that PEPCK and G6PC transcript levels were reduced by >80% in cells treated with bitter melon extract.
Conclusion: Our study indicates that bitter melon may be beneficial to diabetics by reducing their capacity for gluconeogenesis and thereby facilitating the management of blood glucose levels.
| Poster #: 16 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: RNA processing , splicing, protein interactions Project Title: Paralogous RNA binding proteins have distinct partner proteins under neuronal splicing conditions Author List: Sullivan, Michael; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author, Nagel Award Nominee
Edburg, Jacob; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Nunez, Christopher; Graduate, Chemistry and Biochemistry, California State University, Fullerton
Keppetipola, Niroshika; Faculty, Chemistry and Biochemistry, California State University, Fullerton Abstract An important aspect of eukaryotic life is the ability to alternatively splice pre-mRNA into mature mRNA, allowing for more than one mRNA isoform from a gene transcript and in turn protein isoforms. The process of alternative splicing is regulated in part by RNA binding proteins (RBPs) that bind to splice sites on the pre-mRNA and influence the assembly of a spliceosome either positively or negatively at adjacent splice sites. Many of these RNA binding proteins occur as gene families with members sharing high primary structure identity and similar domain organization. Yet, members have tissue specific expression patterns and regulate distinct sets of target exons to exert tissue specific splicing outcome. How related members within a gene family can exert different splicing outcomes is not well understood. One such family of RBPs is Polypyridine Tract Binding Proteins PTBP1 and PTBP2. The paralogs have 74% sequence identity and have high tertiary structure similarity. Changes in the levels of PTBP1 and PTBP2 during neuronal differentiation directs changes in the splicing program critical for the process of neuronal maturation. Understanding how these paralogs can exert different splicing outcomes will give insight to how gene expression is regulated tissue specifically. To this end, in this study we aimed to identify proteins that interact with PTBP1 and PTBP2 in WERI retinoblastoma nuclear extract. Recombinant expressed PTB proteins were incubated in splicing reaction mixtures containing WERI nuclear extract, re-purified and probed for interacting proteins via mass spectrometry. A reaction mixture containing WERI nuclear extract without PTB proteins served as a control. Our results highlight proteins that interact with PTBP1 and PTBP2 as well as those distinct to each paralog. Our results also reveal enzymes and splicing factors unique to each PTBP. Our findings suggest the paralogs have unique protein-protein interactions that likely contribute to their ability to regulate distinct sets of target exons. | Poster #: 17 Campus: California State University, Sacramento Poster Category: Biochemistry Keywords: homology modeling, enzyme inhibition , computational ligand docking Project Title: Computational Analysis of Interactions between Spiroindolones and the Plasmodium Enzyme PfATP4 Author List: Al Chalabi, Linh; Graduate, Chemistry, California State University, Sacramento, Presenting Author
Paula, Stefan; Faculty, Chemistry , California State University, Sacramento Abstract Spiroindolones are considered a new class of antimalarial compounds due to their activity against the parasite Plasmodium falciparum during the blood stage of the disease. The antimalarial activity of spiroindolones stems presumably from their ability to inhibit the enzyme PfATP4, a sodium translocating membrane protein critical for the parasite’s survival. To date, the spiroindolone binding site of PfATP4 and the interactions between spiroindolones and the enzyme remain elusive. This has hampered a structural optimization of this compound class to enhance its properties and ultimately obtain viable antimalaria drug candidates. Here, we employed computational tools to identify the likely spiroindolone/PfATP4 binding site and characterize critical enzyme/inhibitor interactions. We created and validated a homology model for PfATP4 using the crystal structure of the closely related sodium/potassium ATPase as a template. Next, we scanned the surface of the homology model in its transmembrane domain for potential binding sites. The structures of several active spiroindolone compounds were modeled and then computationally docked into the most likely binding site of PfATP4. This facilitated the identification of the most important enzyme/inhibitor interactions and allowed the establishment of a pharmacophore which can be the basis for future virtual screen for more potent inhibitors. | Poster #: 18 Campus: California State University, Stanislaus Poster Category: Keywords: coumarin, toluene monooxygenases, protein engineering Project Title: Metabolism of Coumarin by Toluene Monooxygenases and Engineered Toluene o-Xylene Monooxygenases of Pseudomonas sp. OX1 Author List: Younan, Noella; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Gaddo, Isabel; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Vardar-Schara, Gönül; Faculty, Chemistry, California State University, Stanislaus
Abstract Bacterial toluene monooxygenases (TMOs) are known for their ability to oxidize a wide range of aromatics, degrade a variety of organic pollutants, and produce industrially important compounds. Coumarin, which occurs naturally in many plants, is mostly metabolized into 7-hydroxycoumarin (7-HC) in human liver by P450 monooxygenases. 7-HC, also known as umbelliferone, and other coumarin derivatives have been reported to have various pharmacological and chemo-preventative benefits for human health. Umbelliferone is also used as a sunscreen agent and an optical brightener for textiles due to its strong ability to absorb UV light. In this study, metabolism of coumarin was examined with Escherichia coli strains expressing native TMOs including toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1, toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4, toluene para-monooxygenase (TpMO) of Ralstonia pickettii PKO1, and toluene 4-monooxygenase (T4MO·BTAi1) of Bradyrhizobium sp. BTAi1 for the very first time. Oxidation activity levels and regiospecificities were determined using reverse-phase high-performance liquid chromatography on a C8 column using a water/acetonitrile solvent system. We found that native ToMO successfully metabolized coumarin and produced mixtures of 5-HC, 6-HC, and 7-HC. T4MO·BTAi1 metabolized coumarin into 7-HC, whereas TpMO formed ~70% 6-HC. No metabolites were detected when using the native enzyme TOM. After these reactions were discovered, selected engineered enzymes containing mutations in ToMO were examined in order to generate further improvements. We found that regiospecificity on coumarin has been improved by up to 3.3-fold relative to the native ToMO and the key mutations perturbing ToMO’s active site facilitated human P450-like hydroxylation of coumarin. The results of the present study shows that native and engineered TMOs may be employed for the synthesis of HCs via whole-cell biocatalysis. Furthermore, this study also shows the potential of non-human TMOs and ToMO variants in drug metabolism applications; hence, their applicability to selectively hydroxylate selected drug molecules is currently being investigated. This project is supported by the National Institutes of Health Support of Competitive Research Program. | Poster #: 19 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: Alzheimer’s Disease, Enzyme inhibition, Structure-activity relationship study Project Title: Design, Synthesis, and Biological Evaluation of Donepezil-Based Analogs as Possible Alzheimer’s Disease Therapeutics Author List: Chi, Jasmine; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Dole, Paulina; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Pecic, Stevan; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Abstract Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by memory degradation and loss of cognitive function. Acetylcholinesterase (AChE) is an important enzyme that hydrolyzes the neurotransmitter acetylcholine. According to the Cholinergic hypothesis, AChE inhibitors represent a promising therapeutic target for the treatment of AD symptoms. Donepezil is one of the current FDA-approved drugs that treat AD symptomatically through the inhibition of AChE. However, it can only treat mild-to-moderate AD symptoms and cannot stop the progression of the disease. The long-term goal of our lab is to identify novel drugs to effectively treat AD symptoms. Using structure-activity relationship (SAR) studies, we made chemical modifications of the phenyl moiety of donepezil by placing various fluoro-, chloro-, and methyl-benzyl groups to the indanone-piperidine moiety which is a central pharmacophore core of donepezil. We were able to obtain a library of twenty donepezil-based analogs using a microwave synthesizer and following a reductive amination synthetic procedure optimized in our lab. All analogs were purified and biologically evaluated for their potency against electric eel AChE enzyme. Our SAR study revealed that the modifications on the ortho- and para- positions of the phenyl ring of donepezil resulted in significant AChE inhibition. The most potent analogs showed inhibition in low nanomolar range, similar to donepezil. The preliminary kinetic studies and molecular modeling experiments suggest that these new analogs bind to the enzyme as a mixed-type inhibitor and that fluoro groups are well tolerated in the binding site of the AChE enzyme. Next, we performed several important pharmacokinetic and pharmacodynamic predictions of the most potent analogs and the data obtained suggest that these AChE inhibitors are not toxic, have good metabolic stability and are good drug candidates that can be further explored in the future preclinical studies and the drug design for AD therapeutics. | Poster #: 20 Campus: California State University, Sacramento Poster Category: Biochemistry Keywords: oxidative damage, Aging, Drosophila Project Title: Increased oxidative damage in hyper-aggressive Drosophila melanogaster Author List: al-Rakabi, Arwa; Graduate, Chemistry, California State University, Sacramento, Presenting Author
McInnes, Abigail; Undergraduate, Chemistry, California State University, Sacramento
Bauer, Johannes; Faculty, Chemistry, California State University, Sacramento
Abstract Quality of social environment has been linked to health outcomes and longevity. For example, female baboons with a larger social circle and high-quality social interactions have greater life span than those females with fewer and lower quality social interactions. One important factor influencing the quality of social environment is aggression. Increased aggression/fighting behavior is associated with high stress and extra energy costs that may negatively affect health and longevity traits. For example, fighting behavior is negatively correlated with reduced milk yields in cattle, and higher agreeableness is correlated with longevity in chimpanzees. Data obtained in our lab shows that hyper-aggressive fruit flies experience a poor social environment and have reduced longevity. Together, these data suggest that modulation of aggression behavior impacts aging and longevity.
Here, we use the fruit fly Drosophila melanogaster as a model to investigate the molecular basis of social environment-dependent effects on health and longevity. We focus on the effects of hyper-aggressive behavior by utilizing flies in which the gene Cyp6a20 has been disabled. The function of Cyp6a20 is unknown, but preliminary studies from our lab have shown that flies lacking Cyp6a20 are experiencing a poor social environment, are hyper-aggressive and have reduced longevity.
To confirm changes to the social environment in hyper-aggressive flies, we measured the expression levels of pheromones, hydrophobic molecules on the cuticle of the fly that govern fly behavior. Out data shows that the levels of pheromones involved in aggression and aggregation/group behavior are elevated. These molecular data thus confirm the hyper-aggression behavior of these flies, and moreover suggest that group and social dynamics are altered in these flies.
To understand the molecular nature of these altered group dynamics, we hypothesized that flies living in poor social environments experience higher levels of stress. Stress in molecular terms is oxidative damage to biological macromolecules. Therefore, we measured lipid peroxide content in our hyper-aggressive flies and found it elevated in both male and female flies. We will next asses oxidative damage levels of protein and DNA.
Our data suggests that negative social environments have negative effects on health and longevity. The molecular basis for these effects may be found in increased oxidative stress, resulting in increased macromolecular damage.
| Poster #: 21 Campus: San Francisco State University Poster Category: Biochemistry Keywords: epoxidation, Styrene Monooxygenase, in silico docking Project Title: Substrate, coenzyme, and transition-state analog docking as guide to active site discovery and remodeling for biocatalysis Author List: Ochoa, Biviana; Graduate, Chemistry and Biochemistry, San Francisco State University, Presenting Author
Gassner, George; Faculty, Chemistry and Biochemistry, San Francisco State University Abstract The natural production and industrial synthesis of styrene has increased environmental exposure and contamination; Pseudomonas bacteria have a built-in mechanism that allows them to use styrene as a catabolite and energy source. The enzyme activities of the styrene pathway provide a platform for the development of biocatalysts in biotechnology. Enzyme and biochemical pathway engineering allow for the development of cost-effective and environmentally friendly “green chemical” approaches in the production of fine chemicals. Styrene monooxygenases (SMOs) composed of FAD-specific epoxidase (SMOA) and NADH-specific reductase (SMOB), catalyze the side-chain epoxidation of styrene substrates. This research targets the engineering of the active site of NSMOA, an N-terminally histidine-tagged SMO, which epoxidizes styrene in the aqueous phase with ~99% enantioselectivity. Only the apo-enzyme structure of NSMOA is crystallographically identified. In this study we use in silico docking methods to resolve the substrate and cofactor binding sites of NSMOA to advance our understanding of its specificity and enantioselectivity. The epoxidation reaction of NSMOA proceeds through a nucleophilic attack of the vinyl-side chain of styrene to the terminal oxygen atom of the C(4a)-FAD hydroperoxide intermediate. The enantioselectivity depends on the orientation of the styrene ring with respect to the FAD-hydroperoxide intermediate in the oxygen-transfer reaction. Transition state analogs were prepared with oxygen atoms bridging from the C(4a)-position of the re-face or the si-face of FAD to a range of styrene substrates. The bridged transition state analogs decrease the rotational and translational degrees of freedom associated with individually docking FAD and styrene to NSMOA. The best candidate transition state analog complexes of NSMOA identified in this work are those which bound with high affinity and oriented the substrate in an internal binding pocket. This provides a structural hypothesis that can be evaluated by preparing and characterizing targeted active site mutants. Resolution of the NSMOA active site structure provides a basis for understanding the substrate specificity of SMOs and will contribute to their development as biocatalysts. | Poster #: 22 Campus: California State University, Northridge Poster Category: Biochemistry Keywords: Fluorescence Microscopy, DNA Repair, Yeast Project Title: Creation of a Yen1-RFP strain of S. cerevisiae for Investigation of Yen1 in Double Strand Break Repair Author List: Pineda, Janelli; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Aros, Melissa; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Fischhaber, Paula; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract Double-strand breaks (DSBs) in DNA arise from myriad sources, including those that stall replication, causing mutations in DNA that may lead to cancer and aging. The DSB repair pathway employed varies due to the break location and cell cycle phase. To understand the role of end-processing nucleases in DSB Repair in S. cerevisiae, Red or Yellow Fluorescent Protein (RFP or YFP) tagged versions of Yen1, Mus81, and Rad10 (Yen1-RFP, Mus81-RFP and Rad10-YFP) are being employed to investigate homologous recombination-mediated DSB Repair at anaphase bridges. The localization of Yen1-RFP and Mus81-RFP to anaphase bridges will show whether the structure- specific endonucleases Yen1, Mus81-Mms4 and Rad1-Rad10 participate in resolution of intertwined chromosomes. This project focused on creating an S. cerevisiae strain containing a Yen1-RFP gene.
To achieve the target DNA plasmid needed to create the strain, we used standard cloning techniques in which a Yen1-GFP plasmid was digested and purified to remove the GFP fragment. An RFP-containing fragment from plasmid pNEB30 was then amplified by Polymerase Chain Reaction (PCR) and ligated into the Yen1-containing plasmid to obtain the target Yen1-RFP plasmid. We then isolated the Yen1-RFP fragment and transformed it into S. cerevisiae, to create the target recombinant Yen1-RFP strain. The new strain was screened by PCR and DNA sequencing to confirm the correct integration of Yen1-RFP, showing the presence of the full length gene with no mutations. The Yen1-RFP strain was also characterized by fluorescence microscopy, showing the presence of the expected nuclear-localized RFP signal. Next, the Yen1-RFP gene will be combined with other fluorescently labeled endonucleases by genetic crosses to achieve strains enabling investigation of Yen1-RFP in anaphase bridges by fluorescence microscopy.
We are also investigating by fluorescence microscopy whether Rad1-Rad10 and Mus81-Mms4 are recruited to ultrafine bridges, in response to Zeocin-induced DNA damage using a strain containing Rad10-YFP, Mus81-RFP and Dpb11-CFP. In earlier work, we found that high Zeocin concentrations cause G2/M cell cycle arrest. We are conducting additional experiments to determine whether lower Zeocin concentrations alleviate cell cycle arrest. Together with successful preparation of Yen1-RFP strain, this work will inform our understanding of how end-processing nucleases mediate DSB repair and can reduce mutagenesis and chromosomal instability. | Poster #: 23 Campus: California State University, Bakersfield Poster Category: Biochemistry Keywords: Protein Chemistry, Sulfur Reduction, Archaea Project Title: Exploration of Enzymatic Sulfur Reduction in Fossil Fuels Author List: Al-Azzam, Julnar; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield, Presenting Author
Lopez, Karlo; Faculty, Chemistry and Biochemistry, California State University, Bakersfield Abstract The breakdown of sulfur components in fuels has been, and continues to be, a long and arduous process requiring high heat to break down organosulfur molecules. Early in 2017, Robert Grubb’s group at California Institute of Technology reported a more environmentally friendly way to breakdown sulfur-containing molecules. Their approach uses potassium tert-butoxide triethylsilane to create silyl radicals to break carbon-sulfur bonds and then use silicon to remove the sulfur. This method, which they call KOSi reduces sulfur in diesel fuels to 2ppm, far lower than the current environmental target of 15 ppm. However, the published methodology is a proof-of-concept experiment that has not yet been adapted to an industrial scale, it is designed to work with existing sulfur removal processes, and uses organic reagents to carry out the process. As a result, and notwithstanding this breakthrough, the use of sulfuric acid and high heat remain the process of choice to remove sulfur-containing compounds from fossil fuels. A more environmentally friendly approach to removing sulfur from fossil fuels involves the use of sulfur reducing enzymes, such as Pyrococcus horikoshii disulfide reductase (phCoADR). This enzyme could provide a completely green approach to sulfur reduction, if it couldbe made soluble and retain its activity in an organic medium. This flavin-containing enzyme is robust because it is isolated from a hyperthermophilic Archaea and has been shown to reduce the sulfur by means of a free cysteine residue. Sulfur containing molecules, such as ethanethiol, which are commonly found in petroleum products could be reduced by the enzyme into H2S(g) which can then be collected and precipitated into elemental sulfur or converted into sulfuric acid. Preliminary data has shown that the enzyme can reduce polysulfides with a Kcat of 1.1-18.3 sec-1 depending on the cosubstrate and can be mutated with relative ease. This project focuses on testing and mutating the enzyme so that it can carry out sulfur reduction in organic solvents. | Poster #: 24 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: cardiovascular disease, lipid and lipoproteins, spectroscopy Project Title: Fluorescence Spectroscopic Analysis of Apolipoprotein AI Reconstituted High Density Lipoprotein Author List: Khiev, Kasandra; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Meyer, Kyle; Graduate, Chemistry and Biochemistry, California State University, Long Beach Abstract High density lipoproteins (HDL) (also known as ‘the good cholesterol’) are protein-lipid complexes that aid in cholesterol efflux, a process in which HDL particles interact with ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 to remove excess cholesterol from macrophages. The major protein on HDL, apolipoprotein AI (apoAI) (245 amino acids), has a high helical propensity and can exist in lipid-free and lipid-bound states; its structural flexibility is a signature feature of its lipid (cholesterol) binding functional ability. When bound to phospholipids, apoAI contains 10 alpha-helices (H1-H10) that wrap around the hydrophobic lipid tails. Our goal is to understand how the conformation of apoAI changes during cholesterol efflux using a combination of fluorescence spectroscopy and biochemical cross-linking assays. We hypothesize that residues 125-158 (helices H5 and H6) form a disordered loop that can accommodate lipid loading and changes in HDL particle size. To test this hypothesis, an apoAI double-cysteine mutant (L134C/A152C) was designed with cysteines positioned on the loop and hexa-His tag at the N-terminal end. The protein was overexpressed in E. coli and purified by affinity chromatography. In initial studies, the purified protein was labeled with N-(1-pyrene)-maleimide, a spatially sensitive fluorophore that has a distinct emission at ~460 nm when it is within ~10Å of a neighboring pyrene. The pyrene-labeled apoAI was reconstituted with phospholipids at different phospholipid: protein molar ratios (28:1, 70:1, and 100:1) to generate small (~7.8 nm), medium (~9.6 nm) and large (~10.5 nm) diameter HDL referred to as reconstituted HDL (rHDL). Fluorescence analysis revealed that in the rHDL-bound state, apoAI undergoes conformational reorganization with a significant decrease in excimer emission. The data suggest that the conformational changes accommodate for lipid loading; further studies are in progress to obtain details about the conformational reorganization during cholesterol efflux from J774.1 macrophages using pyrene labeled apoAI in lipid-free state or as small, medium or large rHDL as acceptors. This study is significant since it addresses a critical early step in cholesterol plaque formation from a mechanistic perspective and has direct relevance in cardiovascular disease.
Acknowledgments: This project is supported by the National Institutes of Health (NIH) under the grant number: GM105561 | Poster #: 25 Campus: Sonoma State University Poster Category: Biochemistry Keywords: macrophage, polysulfides, inflammation Project Title: Treatment with Cysteine Trisulfide Induces the Generation of Hydropersulfides and Inhibits the Production of Inflammatory Cytokines Author List: Goddu, Robert; Graduate, Biology, Sonoma State University
Pen, Serena; Undergraduate, Biology, Sonoma State University, Presenting Author
Hanley, Megan; Undergraduate, Biology, Sonoma State University, Presenting Author
Henderson, Catherine; Undergraduate, Biology, Sonoma State University
Fukuto, Jon; Faculty, Chemistry, Sonoma State University
Lin, Joseph; Faculty, Biology, Sonoma State University Abstract The role of hydrogen sulfide in cellular and physiologic systems has been a topic of interest for many decades. While hydrogen sulfide itself has been extensively investigated by several groups, less is known about the effects of the hydropersulfides (RSSH) and polysulfides that can be generated following the treatment of cells with hydrogen sulfide. In this study, we examine the effects of the polysulfide cysteine trisulfide (Cys-SSS-Cys; also known as thiocystine) on mammalian cells. We show that Cys-SSS-Cys treatment of cells results in the rapid production of cysteine hydropersulfide (Cys-SSH). These hydropersulfides display a protective effect against electrophile-induced toxicity. We go on to show that inflammatory cytokine production by macrophages is inhibited in cells treated with Cys-SSS-Cys. These data together demonstrate that cells can utilize Cys-SSS-Cys to generate Cys-SSH and that the Cys-SSH can protect cellular thiols from reactivity with electrophiles and inhibit the inflammatory response. | Poster #: 26 Campus: California State University, Los Angeles Poster Category: Biochemistry Keywords: thermal stability, recombinant protein, cell disruption Project Title: Effects of Thermolysis on the Purification of Recombinant Beetle Antifreeze Proteins Author List: Raminani, Shanti ; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Nguyen, Cindy; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Wen, Xin; Faculty, Chemistry and Biochemistry, California State University, Los Angeles
Abstract Antifreeze proteins (AFPs) are a class of proteins characterized by their ability to inhibit ice-crystal growth and lower the freezing point of bodily fluids, thus leading to the prolonged survival of organisms in subzero temperatures. AFPs derived from the beetles Dendroides canadensis (DAFP-1) and Tenebrio molitor (TmAFP) are commonly expressed in the host bacteria Escherichia coli, enzymatically lysed, and purified. However, this method can be time intensive and cost inefficient. Heat disruption of E. coli cells, referred to as thermolysis, was reported to release heat-stable intracellular enzymes effectively. We hypothesize that thermolysis of the E. coli cells containing the AFPs (i.e., DAFP-1 and TmAFP, which are both thermostable) will release the AFPs more effectively than conventional lysis methods because it will cause native E. coli proteinaceous impurities to denature and/or aggregate. To investigate the thermolysis effects, we treated the cells at variable temperatures (60oC, 70oC, 80oC) and for variable time periods (20, 40, 60 min) and monitored the lysis results using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The results show that temperatures ranging from 60-80oC were able to release most of the AFPs, while remove the most impurities from the cells. Future work will be to incorporate the thermolysis method into the purification process to shorten the purification process and achieve a higher yield of the AFPs for a large-scale production. | Poster #: 27 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: Endoplasmic reticulum stress, Unfolded Protein Response, Akt Project Title: Isoform 2 of protein kinase Akt is a negative regulator of the unfolded protein response Author List: Palma, Miguel; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Perez, Samantha; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Tran, Shayla ; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach
Bhandari, Deepali; Faculty, Chemistry and Biochemistry, California State University, Long Beach Abstract The endoplasmic reticulum (ER) is a cellular organelle that is a site of several important functions including protein folding and secretion. Under conditions that generate ER stress such as imbalance in the protein folding load and capacity, cells begin a signaling program called the unfolded protein response (UPR). UPR serves to restore homeostasis by triggering transcriptional pathways through stress sensing proteins in the ER membrane. In recent years, several groups including ours, have shown that cell survival during ER stress is regulated by a protein kinase known as Akt. Akt is a serine/threonine kinase that plays key roles in many important cellular processes including survival, metabolism, and migration. Although there are three isoforms of Akt (Akt1, 2, 3), most of the studies on Akt use reagents that do not distinguish between the isoforms. While Akt1 and 2 are ubiquitously expressed, expression of Akt3 is restricted to specific tissues. This work aimed to identify the specific Akt isoform that regulates UPR. We used a colorectal adenocarcinoma cell line that only expresses Akt1 and Akt2. In addition to the parental cell line, we included Akt1 knockout, Akt2 knockout and double Akt1/Akt2 knockout cell lines in our experimental design. ER stress was triggered using tunicamycin, which inhibits glycosylation of newly synthesized proteins in the ER thereby promoting their misfolding. Cell lysates were analyzed by western blotting. Using isoform specific antibodies, we determined that while both isoforms showed activation upon induction of ER stress, Akt2 appeared to have a more appreciable increase in activity. Upon fractionating the cell lysates using differential centrifugation, we observed an ER-stress dependent increase in localization of Akt2 to the ER fraction. Finally, our data showed increased signaling output from a specific signaling branch of UPR in Akt2 knockout cell line. Together, these data implied that the activation of Akt2 during ER stress results in negative regulation of UPR. Our future experiments entail investigating unique interactions between Akt2 and UPR effector proteins. | Poster #: 28 Campus: California State University, Chico Poster Category: Biochemistry Keywords: decontamination, ZIF, Lead Project Title: Zeolitic Imidazole Framework (ZIF) Decorated Mucor Hyphae Membranes and Melamine Sponges for Lead(II) Removal From Water Author List: Cash, Angel; Faculty, Chemistry and Biochemistry, California State University, Chico, Presenting Author
So, Monica; Faculty, Chemistry and Biochemistry, California State University, Chico Abstract Lead contamination in water threatens ecosystems and human health. Though activated carbon in Brita filters is widely used in households to remove most water contaminants, they do not remove trace levels (<0.15 ppb) of lead which may lead to chronic health problems. Part of the problem is because activated carbon cannot be synthetically tuned to target specific ranges or types of contaminants. Therefore, we need to develop synthetically tunable filtration materials that can remove trace levels of lead to ensure environmental and public health. Zeolitic imidazolate frameworks (ZIFs), a class of highly tunable and nanoporous hybrid organic-inorganic solids, are potential candidates for water filtration. Since ZIFs are loose powders, mounting them onto membranes allows for easier filtration and retrieval for water decontamination purposes when deployed in the field. While some work has been done on membrane-mounted ZIFs for water filtration, there has been no existing work comparing the effects of different mounting strategies of ZIFs on lead removal in water. In this work, we compare lead decontamination utilizing ZIF-8 and ZIF-67 loose powders, those mounted on melamine foam, and those mounted on Mucor hiemalis hyphae membranes. We assessed the adsorption capacity (qt) and metal leaching of these different ZIF mounting strategies. After exposing all samples to 80 ppm lead solution for 48 hours, we determined the following: (1) Powder ZIF-8 and ZIF-67 reached qt of 309 mg/g and 874 mg/g, respectively. These qt values are comparable to previous work of ZIF-8 and ZIF-67 at an initial lead concentration of 80 ppm. (2) ZIFs mounted onto Mucor membranes had little effect on the lead adsorption but greatly decreased the molar percentage of zinc leaching of ZIF-8 from 59.6% to 0.34% when compared to loose powders of ZIF-8. (3) Mounting ZIFs on the melamine foam decreased their metal leaching but reduced the qt. (4) Data supported ion exchange as a possible mechanism of the adsorption of lead, especially ZIF-67, which had 81% of lead leached into solution and the highest adsorption capacity. Future work will look at improving the stability of ZIF-67 as it was the highest performing in lead adsorption. Based on previous work, doping ZIF-67 with zinc has shown improved water stability. We will be looking at the lead adsorption of ZIF-67 doped with zinc compared to the ZIF-67 that is not doped. | Poster #: 29 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: Cyclin dependent kinase 5, Insulin signaling, Type 2 diabetes Project Title: Cyclin Dependent Kinase 5 Inhibitor Roscovitine Downregulates Insulin Signaling Author List: Castillo, Michelle; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Bhandari, Deepali; Faculty, Chemistry and Biochemistry, California State University, Long Beach Abstract Type 2 Diabetes is a growing major public health concern. Its pathogenesis involves gradual loss of responsiveness to insulin signaling in target tissues such as muscles. This insulin resistance causes dysfunction in glucose uptake from the blood through a pathway that promotes insulin-stimulated translocation of glucose transporter 4 (GLUT4) to the cell surface. The mechanism of GLUT4 translocation is not fully understood, however, previous studies have implicated a role for the cyclin-dependent kinase 5 (CDK5). This project aims to determine the involvement of CDK5 in insulin signaling and GLUT4 translocation in a rat muscle cell line by assessing the effect of CDK5 inhibition on insulin signaling and the extent of GLUT4 translocation. Using western blotting, we monitored insulin signaling in control cells and compared it to the cells that were treated with roscovitine, a competitive CDK5 inhibitor. Our results show that insulin signaling is downregulated in roscovitine treated cells. To track insulin-dependent GLUT translocation to the cell surface in response to insulin, we utilized immunofluorescence microscopy. Our data suggest that GLUT4 translocates to the cell surface within 20 minutes of insulin treatment. To get a quantitative measure of GLUT4 translocation, we plan to employ flow cytometry in our future experiments and compare translocation in control cells to that of roscovitine treated cells. We will also use RNA interference to determine if the changes we observed upon roscovitine treatment are indeed due to CDK5 inactivation. Overall, our results will further our knowledge of the underlying mechanisms of GLUT4 translocation to aid in establishing more effective methods of treatment to relieve the burden of insulin resistance in Type 2 Diabetes. | Poster #: 30 Campus: California State University, Fresno Poster Category: Biochemistry Keywords: Saccharomyces cerevisiae, Microplate-based spectrophotometry, Bcl-2 family proteins Project Title: Growth rate screening of Bcl-2 family proteins expressing yeast by microplate spectrophotometry Author List: Carpenter, Sean; Graduate, Chemistry & Biochemistry, California State University, Fresno, Presenting Author
Gilbert, Andrew; Graduate, Chemistry & Biochemistry, California State University, Fresno, Presenting Author
Dejean, Laurent; Faculty, Chemistry & Biochemistry, California State University, Fresno
Abstract Due to their use as biological models and their potential for industrial application, microorganisms such as bacteria and unicellular fungi are of great interest to researchers. From genetic engineering to drug development, determination of the growth rate of these organisms is often the foundation of many areas of microbiological research. The most common method used to calculate the growth rate of microorganisms is to measure the optical density of cells at 600 nm (OD600) using a traditional UV-Vis spectrophotometer. If the OD600 is proportional to the number of cells in solution at a given time, it stands to reason that monitoring this value as the cells grow can be used to estimate how quickly they are dividing. Despite being the gold-standard method for assessing microbial growth, measuring the OD600 in this manner has several limitations. For example, manual collection of absorbance data removes the cell culture from optimal growing conditions for the duration of the collection period, which may impede cell growth. Furthermore, this method has considerable logistics concerns, as data collection can take several hours with repeated measurements every 30-60 minutes. Here, we present a novel, high throughput approach to OD600-based growth rate calculations using a 96-well plate format. The diploid Saccharomyces cerevisiae strain W303-2n (mat α, ade1/ade1, his3/his3, leu2/leu2, trp1/trp1, ura3/ura3) was transformed to express bcl-2, a human proto-oncogene using the pCM184 shuttle plasmid. The growth rate on a minimal liquid culture glucose media of this mutant was compared to that of a vector control construct lacking the bcl-2 transgene. In the 96-well plate format, the Bcl-2 expressing yeast displayed a doubling time of 3.07 ± 0.05 hours (avg. ± SEM, n=9), while the pCM184 vector control yeast were calculated to have a doubling time of 3.15 ± 0.06 hours (avg. ± SEM, n=9). Although these experiments did not show any significant difference in doubling times between the Bcl-2 expressors and control (p=0.341, α=0.05), the data suggest that microplate spectrophotometry is an effective and reproducible method of determining the growth rate of microorganisms cultivated in liquid media.
This research has been funded by CSU, Fresno: College of Science and Mathematics Faculty Sponsored Research Awards (FSSRA) to SC and AG | Poster #: 31 Campus: San Diego State University Poster Category: Biochemistry Keywords: Biomaterials , Spider Silk, NMR Project Title: Isotope Enriched Spider Silk Proteins for NMR Structure Studies Author List: Chau, Andy; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Domingo, Anikin ; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Johnson, Hannah; Graduate, Chemistry and Biochemistry, San Diego State University
Chalek, Kevin; Postdoc, Chemistry and Biochemistry, San Diego State University
Onofrei, David; Staff, Chemistry and Biochemistry, San Diego State University
Holland, Gregory; Faculty, Chemistry and Biochemistry, San Diego State University Abstract Invention inspired by nature is not a new concept, from cars modelled after boxfish to Velcro designed from plant burrs natural inspiration is found throughout modern technology. The study of biomaterials has opened the door to creating new and improved materials using biopolymers found in nature as design models. One biomaterial that has been extensively studied by our lab is spider silk. Stronger than steel and tougher than Kevlar, the material used to fabricate bullet proof vests, spider silk has potential for a wide range of biotechnology applications from bone grafting to suturing materials. The problem, however, is synthetic spider silk fibers spun from recombinant proteins fall short of their native counterparts. To mimic spider silk’s evolutionarily optimized spinning process and create fibers with comparable mechanical properties, we must first elucidate the molecular level structure and intermolecular interactions of the proteins that comprise the starting material (spider silk dope) and the final spun fiber.
Using nuclear magnetic resonance (NMR), we can study atomic-level interactions in spider silk dope and the final fiber format. Certain amino acids, namely arginine, threonine, and leucine, are present in spider silk in relatively low amounts. To determine the role of these sparce amino acids in spider silk assembly, we have developed isotope labeling schemes by feeding spiders 13C/15N-labeled amino acids in their water supply to isotope label and investigate these amino acids in spider silk assembly through NMR spectroscopy. Using 3D solution NMR, we have already confirmed the arginine assignment, which alongside glycine and tyrosine, are notoriously difficult to identify in the 15N NMR HSQC spectra. Investigating these amino acids in spider silk materials is leading to a better understanding of spider silk structure and assembly. Discovering how spider silk develops from disordered proteins in a hydrogel into β-sheet rich fibers is key to recapitulating this process in the laboratory.
| Poster #: 32 Campus: San José State University Poster Category: Biochemistry Keywords: Drosophila, intracellular pH, developmental biology Project Title: Identifying Proteins that Mediate Cellular Behaviors in Response to Higher Intracellular pH Author List: Martins, Laura; Graduate, Biological Sciences, San José State University, Presenting Author
Wong, Ramy; Undergraduate, Biological Sciences, San José State University, Presenting Author
Orozco, Daniel; Graduate, Biological Sciences, San José State University
Grillo-Hill, Bree; Faculty, Biological Sciences, San José State University Abstract Background: Intracellular pH (pHi) is tightly regulated by cells, and emerging evidence suggests that regulated pHi dynamics modulate distinct cell behaviors. Previous studies demonstrated that a transient increase in pHi is required for cell proliferation across species from yeasts to humans, and constitutively increased pHi is a conserved characteristic of cancers. The sodium-proton exchanger NHE1 is a key regulator of pHi, and has increased expression or activity in many cancers. To study the role of dysregulated pHi in cancer, we generated transgenic flies that over-express the homolog of NHE1, DNhe2 (OE-DNhe2), which is sufficient to increase pHi and increase cell proliferation. Our objective is to identify the proteins that promote increased proliferation in response to higher pHi using unbiased genetic screens.
Methods: We screened a collection of 193 Drosophila lines covering 94% of the second chromosome. We visually inspected flies for enhancement or suppression of the OE-DNhe2 rough eye phenotype. We identified 35 regions of the second chromosome that show a genetic interaction with OE-DNhe2. We focused on a region identified by two overlapping deficiencies (Df (2L)ED1303 and Df(2L)ED1315 spanning 38B4-38C6) that both suppressed the OE-DNhe2 rough eye phenotype.
Results: We identified 8 candidate genes in the region defined by the overlapping deficiencies. We obtained genetic reagents to alter expression of each gene, and tested them for suppression of OE-DNhe2. Two of these genes, CG10949 and CG31688, showed genetic interactions with predicted loss-of-function alleles. For the gene CG10949, both RNAi and P-element alleles showed strong suppression of the OE-DNhe2 rough eye. For the gene CG31688, we obtained P-element and Minos transposable insertion alleles. The P-element allele showed mild suppression of the OE-DNhe2 rough eye, however the Minos allele showed no interaction with the OE-DNhe2 rough eye. CG10949 consistently demonstrated strong suppression while interaction with CG31688 were variable, therefore future efforts will focus on characterizing the interaction between OE-DNhe2 and CG10949. Next, we will characterize the phenotypes of these genetic interactions to understand the cellular basis of the observed phenotypic suppression. Understanding the effects of increased pHi and pH sensitive proteins that induce hyperproliferation can help us understand and discover new therapeutic targets.
Funded by NIH SC3GM132049 and CSUPERB NI Award
| Poster #: 33 Campus: San Diego State University Poster Category: Biochemistry Keywords: Biomaterials , Spider Silk, Simulations Project Title: Predicting the Three-dimensional Structure of Spider Silk Proteins with AlphaFold and Molecular Dynamics Simulations Author List: Aldana, Julian; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Chalek, Kevin; Postdoc, Chemistry and Biochemistry, San Diego State University
Onofrei, David; Staff, Chemistry and Biochemistry, San Diego State University
Holland, Gregory; Faculty, Chemistry and Biochemistry, San Diego State University Abstract Spider silk is remarkable structural material that exhibits a combination of high tensile strength and extensibility that make it one of the toughest materials known. Scientists have been investigating spider silk as a model system for the design of high-performance materials in biotechnology including our laboratory. The silks are spun in an aqueous environment at ambient temperature and pressure, making it a highly desirable route for sustainable biomaterials production. However, spider silk proteins present significant challenges for understanding their 3D molecular structure as they are generally large (3,000-4,000 amino acids), highly repetitive and intrinsically disordered or contain intrinsically disordered domains (IDR’s) in solution. Intrinsically disordered proteins (IDPs) are a family of proteins that are incredibly challenging to study using traditional techniques like X-ray crystallography (XRD). Structural proteins that form biomaterials (e.g. spider silk proteins) are IDPs or contain IDRs prior to their solidification into fibrous form (silk) making an understanding of the IDP state a critical component to this research.
Improving our current understanding of IDPs is crucial to unravel the molecular mechanisms of biomaterial formation for systems like spider silk. Proteins with these characteristics need to be approached in a way that can allow traditional methods to build upon them, without the complete abandonment of already established techniques. In the present work, we are using AlphaFold to predict the 3D structure of spider silk proteins from their primary amino acid sequence in combination with coarse grain molecular dynamics (MD) simulation using the Martini Force Field Model in the GROMACS simulation package. With the combined use of computational simulation and predictive modeling, together with experimental NMR studies on isotope enriched proteins, we are moving closer to the end goal of determining the 3D structure of silk proteins. This understanding can then be used to hypothesize how multiple proteins come together and assemble into silk fibers.
| Poster #: 34 Campus: California State University, Northridge Poster Category: Biochemistry Keywords: NMR spectroscopy, chaperone protein, protein folding Project Title: Hydrophobic interactions and hydrogen bonds are key to the chaperone activity of HdeB with client proteins Author List: Nguyen, Hiep; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Mead, Nicholas; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Crowhurst, Karin; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract HdeB is an acid-stress chaperone that plays a crucial role in the survival of pathogenic bacterial by protecting other bacterial (“client”) proteins from aggregation and facilitates subsequent refolding upon pH neutralization. HdeB is active and highly dynamic near pH 4. Since HdeB doesn’t undergo significant structural changes at pH 4, it is thought that its function derives from its intrinsic flexibility. A deeper understanding of how HdeB functions is required to disrupt the chaperone’s activity and mitigate the harm caused by those pathogenic bacteria, which cause dysentery.
To investigate the function of HdeB, the client protein ModA was specifically chosen due to its small size, making it suitable for NMR spectroscopy. We probed the interaction between HdeB and ModA with 1H-15N correlation NMR experiments (1H-15N HSQCs). For our first set of experiments our spectra monitored HdeB, which was labeled with the NMR-active isotope 15N; each peak in this spectrum represents a backbone amide for each residue in HdeB. By monitoring chemical shift changes in the HSQC spectra of 15N-labeled HdeB by itself (apo) compared to a mixture of HdeB and ModA (holo) at pH 4.5 we were able to determine which HdeB residues participate in client binding. Spectral peaks for the holo sample also exhibited decreased intensity compared to apo. Both sets of data confirm there may be binding between HdeB and ModA in the space between the dynamic loop (residues 31-43) and the C-terminus of HdeB. To determine whether this site of interaction is universal or specific to each client, we recorded an HSQC spectrum of 15N-labeled HdeB with another client protein, HisJ. Comparison between the spectra of HdeB in the presence of the two client proteins showed that the interaction sites are largely the same.
We also completed the chemical shift assignment of 15N,13C-labeled ModA, which allowed us to characterize which residues in the client interact with HdeB. ModA residues with significant chemical shift changes from apo to holo (with HdeB present) consist largely of amino acids that are hydrophobic or have side chains that can form hydrogen bonds; these are the same type of amino acids that exhibit significant chemical shift changes in HdeB. Future experiments will include adding HdeA, a partner chaperone of HdeB that functions at a lower pH range, to characterize the potential cooperativity between the two chaperones as the bacteria passes through the acidic stomach environment. | Poster #: 35 Campus: California State University San Marcos Poster Category: Biochemistry Keywords: Protein purification , Functional amyloid, Chemical Denaturants Project Title: Protein CsgA From Salmonella Typhimurium Can Be Purified Using Either 3 M Guanidine Hydrochloride or 6 M urea Author List: Alla, Madusudhan ; Undergraduate, Chemistry & Biochemistry , California State University San Marcos, Presenting Author
Gomez, Cristal; Undergraduate, Chemistry & Biochemistry , California State University San Marcos, Presenting Author
Jayasinghe, Sajith; Faculty, Chemistry & Biochemistry , California State University San Marcos
Abstract Salmonella Typhimurium's ability to adhere to a diverse set of surfaces is the result of cell-surface filaments known as Curli fibers. Assembly of Curli requires six different proteins known as CsgA, CsgB, CsgC, CsgE, CsgF, and CsgG. While five of these six proteins are responsible for the transportation of and attachment to the bacterial cell surface, Curli fibers are formed from the aggregation of multiple CsgA monomers. We are interested in understanding the structure of Curli and the molecular details that lead to protein aggregation into curli fibers. To obtain protein in support of this goal we expressed and purified CsgA under denaturing conditions. We expressed C-terminally histidine-tagged CsgA using BL21(DE3) bacteria cells that were transformed with a pET21a plasmid containing the gene for CsgA. Protein purification was carried out using immobilized metal (nickel) affinity chromatography under denaturing conditions using either 3M Guanidine Hydrochloride or 6M urea. After desalting, the purity of the protein was checked using SDS-PAGE. The identity of the protein was confirmed using a western blot. The tendency of CsgA to aggregate makes purification challenging. In this work, we were able to purify CsgA using denaturing conditions. Traditionally Guanidine Hydrochloride is a preferred denaturant; however, it was questioned if Urea would be an appropriate denaturant for the purification of CsgA. It was found that both Guanidine Hydrochloride and Urea were effective in preventing protein aggregation and facilitating purification. Given the lower cost of urea and chargeless nature, we will be conducting future purifications starting at 8M urea, but lower concentrations will be examined.
| Poster #: 36 Campus: California State University San Marcos Poster Category: Biochemistry Keywords: Curli, CsgC, Purification Project Title: Removing the two native cysteine residues does not change the secondary structure of Salmonella CsgC. Author List: Aljayyousi, Zaina ; Undergraduate, Biology, California State University San Marcos, Presenting Author
Molina Smith, Paul; Undergraduate, Chemistry/ Biochemistry, California State University San Marcos, Presenting Author
Jayasinghe, Sajith ; Faculty, Chemistry/ Biochemistry, California State University San Marcos
Abstract Background:
Curli are hair-like protein filaments found on gram-negative bacteria such as
Escherichia and Salmonella that act as extracellular functional amyloids. Curli are
known to function in cell aggregation, host colonization, bacterial motility. Curli
assembly involves six proteins from the curli specific genes (Csg) A,B, C, E, F and G.
Proteins Csg C,E,F, and G build an efficient secretion and chaperone system that
prevents the major structural proteins of curli; CsgA & B from forming intracellular
amyloid aggregates that are toxic to the host. The main goal of our research is to
determine the structure and function of proteins involved in Curli assembly and this this
work is focused on the expression and purification of CsgC to determine structural and
functional characteristics of the protein.
Methodology:
C-Terminally histidine tagged wild type Salmolenella CsgC, Salmonella CsgC lacking
the two conserved native cysteine residues, and E. Coli CsgC we expressed using the
BL(21)DE3 bacterial expression system. Proteins were purified using Nickel affinity
chromatography and desalted using a PD-10 desalting column. After protein
concentration the purity of the sample was checked using SDS- PAGE and its identity
confirmed by Western Blot using a C-terminal anti-his antibody conjugated with horse
radish peroxidase.
Results and Conclusion:
All three proteins were successfully expressed. We obtained circular dichroism spectra
of wild type Salmonella CsgC in the absence and presence of TCEP, a reducing agent,
and did not observe any overall secondary structure changes. Both the wild type and
the cysteine less version of Salmonella CsgC exhibited CD spectra with a prominent
negative ellipticity centered at 218 nm, indicating that the proteins contain significant
beta-sheet character. The lack of change in secondary structure in the presence
excess TCEP suggests that the disulfide bond is not needed for maintaining the
structure of the wild type Salmonella CsgC protein. The circular dichroism spectrum of
E. Coli CsgC, which has approximately 70% sequence similarity to Salmonella CsgC,
also exhibited negative ellipticity at 218 nm and was identical to the spectrum of
Salmonella CsgC. We will use these proteins to determine their ability to prevent the
aggregation of the CsgA, the main Curli structural protein, along with other amyloids
unrelated to Curli. | Poster #: 37 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: Alzheimer’s Disease, Butyrylcholinesterase, Enzymes Project Title: Synthesis and Biochemical Evaluation of Fmoc-Amino Amide Analogs as Butyrylcholinesterase Inhibitors for the Treatment of Alzheimer’s Disease Author List: Khanbabaei, Serli; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Schwans, Jason; Faculty, Chemistry and Biochemistry, California State University, Long Beach Abstract Alzheimer’s Disease (AD) continues to affect millions of people worldwide. While there’s no cure for AD, multiple approaches are being developed. One of these approaches involves cholinesterases, enzymes involved in neuronal signaling, which have been a target due to the changes in activity observed in individual with AD. The enzymes hydrolyze the substrate, acetylcholine, a cationic neurotransmitter. Butyrylcholinestrase (BChE) is one of the two major classes of cholinesterases that are found in the parasympathetic nervous system. Previous studies have reported that BChE activity has been upregulated in individuals with AD. We previously found that amino acid analogs bearing a 9-fluorenylmethyloxycarbonyl (Fmoc) group serve as selective inhibitors of BChE. Although Fmoc amino acids inhibit BChE, their negatively charged carboxylate group could be unfavorable in binding an enzyme that recognizes a cationic substrate. To test this hypothesis, neutral Fmoc-bearing amino amides were synthesized and biochemically evaluated. The amino amides are readily synthesized in one step from the corresponding amino acid on the 100 mg scale, purified by silica gel column chromatography, and characterized by NMR. The IC50 and KI values (inhibition constants) were obtained using colorimetric enzyme kinetics assays monitored by UV-Vis absorbance. Our results showed that all Fmoc-amino amides tested inhibit BChE. Comparing the amide to the corresponding carboxylate showed the largest difference for Fmoc-Leu-NH2 where the KI value of 18 uM was ~six-fold lower than the KI value of 120 uM for the carboxylate. Fmoc-Val-NH2 was an approximately two-fold better inhibitor than the carboxylate. The results support the model that the negative charge of the carboxylate is unfavorable for binding. Building on these results, a more extensive series of amino amides are currently being investigated for their selectivity, reversibility, and type of inhibition. Together, the results are aimed at dissecting the features important for inhibition and furthering our use of Fmoc-amino acid scaffolds for the development of potent cholinergic inhibition and serving as a potential treatment of AD. | Poster #: 38 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: 2,6-diarylidene cycloalkanones, Akt, protein kinase Project Title: Synthesis and analysis of 2,6-diarylidene cycloalkanones as Akt activators Author List: Husain, Aida; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Cardona, Daniel; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Izarraras, Kimberly; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach
Bhandari, Deepali; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Salzameda, Nicholas; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Abstract Serine/threonine protein kinase Akt has over 100 known substrates and is involved in regulation of several important cellular functions including proliferation, survival, metabolism and migration. Due to its role in key cellular processes, it is a promising therapeutic target for diseases such as cancer and type 2 diabetes. As such, there is great interest in the development of regulating Akt activity by small molecules. The focus of this project is to investigate 2,6-diarylidene cycloalkanones as a novel scaffold for modulation of Akt activity. Originally, 2,6-di-(4-pyridyl)methylidene cyclohexanone was reported to cause cell death by allosterically regulating the kinase activity and promoting degradation of Akt. However, our cellular data collected using western blotting based signaling analysis contradicts this model and suggests that Akt is in fact activated upon treatment with this compound. Interestingly, when we replaced the cyclohexanone moiety with cyclopentanone, Akt activation was not observed. However, when the diaryl groups were varied, Akt activation was maintained. Our working theory is that the conjugation between the carbonyl and the aromatic rings along with the cyclohexanone core are vital for Akt activation. To further study the mode of action of this scaffold, we will synthesize a library of 2,6-diarylidene cycloalkanones containing both symmetrical and asymmetric analogs and various substituents on the aromatic rings via Aldol condensation reactions. We will also perform direct binding studies with Akt and in vitro activity assays. This study will help elucidate the structural elements required for Akt activation by 2,6-diarylidene cycloalkanones and further evaluate the potential of these small molecules as Akt activators. | Poster #: 39 Campus: California State University, San Bernardino Poster Category: Biochemistry Keywords: Vesicular transport, Organelle, Cell signaling Project Title: Yeast cells lacking Rab GAPs Gyp2 and Gyp7 suffer Golgi-endosome cargo retrieval defects Author List: Tambunan, Rebecca; Graduate, Biology, California State University, San Bernardino, Presenting Author
Nickerson, Daniel; Faculty, Biology, California State University, San Bernardino Abstract Eukaryotic cells have membrane-bound organelles that perform specialized biochemical functions and depend on vesicular transport for delivery of cargo proteins and lipids to correct compartments. Rab GTPases cycle between active GTP-bound and inactive GDP-bound states in order to regulate timing and specificity of membrane fusion events. Extended vesicular transport pathways, such as the secretory and endocytic, depend on the sequential functions of multiple Rab GTPases. Rabs exhibit slow intrinsic rates of GTP hydrolysis and depend upon GTPase activating proteins (GAPs) as negative regulators of their signaling and localization. However, the identification of specific roles for Rab GAPs in transport pathways is often hindered by functional redundancies among Rab GAPs.
Saccharomyces cerevisiae, Brewer’s yeast, is a unicellular fungus species widely used as a model organism for research in vesicular trafficking due to ease of genetic modification and deep toolkit of biochemical assays. In a genetic screen for defective endolysosomal cargo transport in yeast lacking multiple Rab GAPs, we identified a synthetic defect of a lysosomal enzyme mistargeted to the cell surface in cells lacking both Gyp2 and Gyp7, which are previously characterized to regulate Rab functions at the Golgi and lysosome/vacuole, respectively. We observed both Gyp2 and Gyp7 at endosomal compartments. While the lipid-binding GRAM domain of Gyp2 was previously reported to be required for its function in regulating Rab activity at the Golgi (Sciorra et al., 2005), GRAM function was dispensable for Gyp2 function in endolysosomal transport.
Secretion of lysosomal enzymes is often indicative of either an anterograde or retrograde defect in transport between the Golgi and endosome compartments. Using fluorescence microscopy, we observed that the typically Golgi-localized probe Vrg4-GFP mislocalized to the endolysosome in cells lacking both Gyp2 and Gyp7, indicative of a failure to retrieve Vrg4-GFP from the endosome to return to the Golgi. These data suggest that besides their known roles regulating Rab activity at the Golgi and vacuole, respectively, Gyp2 and Gyp7 cooperate to regulate transport between Golgi and endolysosomes, particularly retrograde transport from the endosome to Golgi.
This work was supported by CSUSB Office of Student Research supplies grant (to RT), a CSUPERB Graduate Student Research Restart award (to RT), and a NIH SC2 GM140979 award (to DPN). | Poster #: 40 Campus: California State University, San Bernardino Poster Category: Biochemistry Keywords: Falcilysin, Malaria, Inhibitor Project Title: Development and Optimization of Selective Click Probes to Investigate the Function of Falcilysin Author List: Bhaskara, I Gusti Ngurah Raka; Undergraduate, Chemistry and Biochemistry, California State University, San Bernardino, Presenting Author
Zavala, Rey; Undergraduate, Chemistry and Biochemistry, California State University, San Bernardino, Presenting Author Abstract Malaria is a fatal parasitic disease that causes approximately 600,000 deaths annually. We are developing small molecule inhibitors as chemical probes to further explore the function of Falcilysin (FLN) - a malarial metalloprotease found in the malaria parasite Plasmodium falciparum - that is crucial for parasitic development in humans. These small molecule inhibitors will block FLN, allowing us to study its role in the parasite’s life cycle and evaluate its potential as a therapeutic drug target.
We synthesized a linker compound that incorporates both a photocrosslinkable group and a click chemistry ‘handle’ to validate whether or not the inhibitor binds exclusively with FLN. When activated by UV light, the photocrosslinkable group will covalently bond the inhibitor to the target protein. A click chemistry ‘handle’ consisting of an alkyne group allows the inhibitor protein complex to be isolated from the treated parasite culture. We can utilize these mechanisms to evaluate the protein targets that bind with the inhibitor.
We synthesized new piperazine-based hydroxamic acid inhibitors that will also incorporate photo-crosslinking and click chemistry moiety, which will allow us to isolate our chemical probes' cellular targets. Our scaffolds include the piperazine-based hydroxamic acid with a 4-biphenyl sulfonyl substituent at the N4 position and 3-bromophenyl sulfonyl and 4-bromophenyl sulfonyl as substituents at the N1 position. This will allow us to quickly introduce a variety of aryl substituents at the N1 position by a Suzuki coupling reaction. Importantly, this will also allow us to attach the newly synthesized linker, which enables click chemistry and photo-crosslinking.
| Poster #: 41 Campus: California State Polytechnic University, Pomona Poster Category: Biochemistry Keywords: copper nitrite reductase, Inorganic chemistry, denitrification Project Title: Computational Analysis of a Copper Containing Nitrite Reductase Using X-ray Emission Spectroscopy Author List: Gorney, Sage; Undergraduate, Chemistry & Biochemistry, California State Polytechnic University, Pomona, Presenting Author
Stieber, S. Chantal E.; Faculty, Chemistry & Biochemistry, California State Polytechnic University, Pomona Abstract Denitrification is a critical component in the nitrogen cycle involves reduction of NO3- and NO2- to N2 and O2. All stages of denitrification are catalyzed by complex metalloenzymes inside soil bacteria, each with their own transition metal cofactors. The catalysts used during nitrite reduction to nitric oxide are nitrite reductases (NiRs). There are two main types of NiRs, one with an iron cofactor (cd1NiRs) and the other with copper (CuNiRs). The mechanisms of CuNiRs are currently not well-understood, partly due to the challenge of probing reactivity at metal centers inside large enzymes. Recent computational work reports possible intermediates in NO2- reduction by CuNiRs, however it is lacking experimental corroboration. X-Ray Emission Spectroscopy (XES) is a element-specific technique that has potential to be used to probe electronic structures and coordination environments of CuNiR. In XES, X-ray photons bombard a sample, exciting a 1s core electron and leaving a hole for the other electrons to fill. When electrons relax to fill a lower energy level, they give off a certain amount of measurable energy, which is what is detected in an XES experiment. Typical ranges for XES spectra for copper is in the region of 8010-8070 eV, and DFT calculated XES have shown good agreement with experimental data. Tthe goal of this research is to computationally probe the potential of XES to distinguish proposed intermediates in NO2- reduction. DFT calculations were conducted for 8 proposed states of the active site in CuNiR. Calculated XES spectra are distinct for each of the 8 states, suggesting that experimental XES could be used to distinguish these. Orbital contributions to XES signals showed that shifts in certain signals could be attributed to changes in the nitrite/nitric oxide binding conformations. Overall, these computational results suggest that XES could be used to experimentally distinguish intermediates in CuNiR. | Poster #: 42 Campus: California State University, Northridge Poster Category: Biochemistry Keywords: G protein selectivity, GPCRs, Molecular dynamics Project Title: Structural Characterization of the G12 Selective Designer Receptor Exclusively Activated by Designer Drugs in Complex with G12 and G13 Proteins Author List: Cruz, Christian; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Inoue, Asuka; Graduate School of Pharmaceutical Sciences, Tohoku University
Abrol, Ravinder; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract G-protein-coupled receptors (GPCRs) are the largest superfamily of integral membrane proteins that are activated by extracellular ligands and enable intracellular signaling pathways through coupling to G-proteins and 𝛃-arrestins. DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) are a chemogenetic tool that are activated by synthetic ligands and can be used to characterize and control G-protein signaling. There are currently DREADDs available that selectively couple to Gs, Gi/o, Gq/11 and G12 proteins. The most recently discovered G12 selective DREADD does not couple to the highly homologous G13 protein. The foundation of this work stems from a collaboration with Dr. Inoue and his paper Illuminating G-Protein-Coupling Selectivity of GPCRs (Inoue et al., 2019 Cell 177, 1933). We are structurally characterizing the G12 selective DREADD (DRE12) in complex with G12 protein (DRE12:G12) and G13 protein (DRE12:G13) using homology modeling followed by molecular dynamics (MD) simulations to relax the complex structures in the membrane environment. MD simulations involved Gaussian Accelerated Molecular Dynamics (GaMD) to conduct enhanced conformational sampling using a harmonic boost potential to reduce energy barriers. Clustering with CPPTRAJ allowed for the diverse conformations that are present most often for the duration of the GaMD simulations to be further analyzed. These simulations allow for analysis of the protein residues and structural motifs of the receptor and G-protein as they interact with one another and the lipid bilayer. Trajectories of the DREADD-G-Protein complex snapshots have been generated to provide insight into the factors that influence the G12 selectivity and lack of G13 selectivity. The total free binding energy between the receptor and G-protein are calculated through MMPBSA (Molecular Mechanics Poisson-Boltzmann Surface Area) method, which has identified that the DRE12:G12 total free binding energy is lower than that for the DRE12:G13 complex. Specific residue interaction analysis is currently being performed through Native Contact Analysis to identify both similarities and differences that play a role in the DRE12’s G12 selectivity to assist in the design of a G13-selective DREADD (DRE13) and enable characterizing G13 signaling in different physiological contexts. Research is funded through BUILD PODER NIH Research fellowship grant #5TL4GM11897707 and #SRL5GM118975-07 as well as in part by NIH grant SC2GM130480. | Poster #: 43 Campus: California State University, Northridge Poster Category: Biochemistry Keywords: Biochemistry, DNA Repair, Affinity Capture Project Title: Investigating the Binding Domain between Rad1 and Slx4 Proteins in the Yeast DNA Double-strand Break Repair Pathway Author List: Chavez, Sarah; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Odango, Jane; Graduate, Chemistry and Biochemistry, California State University, Northridge
Buo, Sorinna; Staff, Chemistry and Biochemistry, California State University, Northridge
Fischhaber, Paula; Faculty, Chemistry and Biochemistry, California State University, Northridge Abstract Double-strand breaks in DNA result from myriad DNA damaging agents deriving from both exogenous and endogenous sources. Since this damage can be lethal, our cells have developed pathways to repair damaged DNA. Double-strand breaks can be repaired through many pathways, one of which is known as single-strand annealing. In single-strand annealing, the double-strand break is located between two DNA repeat sequences. Since the repeat sequences are homologous, the broken ends can be annealed via complementary base pairs on opposite strands, which enables repair of the break, but results in loss of DNA originally lying between the repeats. In the yeast, S. cerevisiae, Rad52 promotes the annealing step and the Rad1-Rad10 endonuclease complex is recruited by Slx4 to cleave the flaps. Following flap cleavage, DNA polymerase fills in the remaining gaps and DNA ligase seals the nick in the phosphodiester backbone. The literature shows that, in humans, the helicase-like domain (HLD) of XPF (yeast Rad1) interacts with the “MLR” domain of Slx4, but analogous interactions have not been carried out in yeast. In this project we used an affinity capture assay to investigate the binding between Rad1 and the MLR domain within Slx4.
To do this we have expressed just the MLR domain of Slx4 as a 6xHIS-tagged protein in E. coli, and have separately co-expressed the full length Rad1-Rad10 complex with an S-tag on Rad1 and a GST-tag on Rad10. Immunoblots from affinity capture experiments carried out with Ni-NTA beads show pulldown of S-tag-Rad1 in the presence 6xHIS-Slx4, but no pulldown in controls lacking Ni-NTA and Slx4. These data suggest that the MLR domain of Slx4 mediates binding and recruitment of Rad1-Rad10 to Double-stand Break Repair sites. | Poster #: 44 Campus: California State University, Fullerton Poster Category: Keywords: pregnancy and inflammation, blood plasma ceruloplasmin, transcription Project Title: Increases in the blood plasma Cu protein, ceruloplasmin, induced by estrogen/progesterone vs inflammatory cytokines: role of transcription Author List: Sanqui, Cossette; Undergraduate, Chemistry and Biocemistry, California State University, Fullerton, Presenting Author
Savage, Cannon; Graduate, Chemistry and Biochemistry, California State University, Fullerton
Linder, Maria C; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Abstract Ceruloplasmin (Cp) is a multifunctional protein, accounting for 40-70% of the total Cu in blood plasma. Circulating levels increase in pregnancy, when taking estrogenic or progestegenic contraceptives, and effects of inflammatory cytokines. The increase in mid-pregnancy reflects a need for Cp to deliver Cu to the placenta and developing fetus. The increase in inflammation is part of the acute phase response, and may protect the surfaces of normal cells from oxidative damage induced by ROS release as part of our immune defense. Earlier rat studies with estrogen showed Cp to increase slowly (2-fold in 2 wks) accompanied by some increase of Cp mRNA in the liver, the main source of circulating Cp. We have now extended these studies to determine whether estrogen also affects expression of the “pump” (ATP7B) that provides Cu to Cp during its production, and to investigate whether the kidney Cp and ATP7B expression might also be affected. We done similar investigations the increase of Cp caused by inflammation, using turpentine model, which has a more rapid effect. Additional confirmatory experiments were done in cultured liver cells.
Female Sprague Dawley rats, 6-8 weeks of age, were injected subcutaneously either with 17-estradiol daily for 2 wks (1 µg/g BW) or with a single injection of turpentine oil (1 µl(/g BW) and were later euthanized for collection of blood and tissues. Pregnant rats were euthanized at 15 d of gestation. Cultured rat hepatoma cells were treated with estradiol for 1 wk before analysis. Cp levels were monitored by measuring Cp oxidase activity with o-dianisidine. mRNA levels were determined relative to 45S rRNA using Real Time qPCR, after RNA extraction and RT.
Treatment with turpentine resulted in a 3-fold increase of Cp in the circulation by 48 h, preceded by 3.5- and 5-fold rises in liver and kidney Cp mRNA, by 16 and 24 h respectively. Although 2-wk estrogen treatment increased circulating Cp 80% there was no significant change in Cp mRNA levels in either liver or kidney. Similar results and changes were observed in pregnancy. Estrogen treatment of cultured hepatic cells also failed to increase Cp mRNA. Steroid and turpentine treatments had little effect on ATP7B mRNA expression. These results provide evidence that kidney and liver respond to inflammation by increasing Cp expression and probably do so by increasing rates of transcription, while the steroid hormones increase circulating Cp levels by post-transcriptional means.
| Poster #: 45 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: , Actin, drebrin, formin Project Title: INTERACTION INTERFACE BETWEEN NEURONAL DREBRIN AND DIA2 FORMIN: POTENTIAL MECHANISM OF DENDRITIC SPINE MATURATION Author List: Srapyan, Sargis; Graduate, Chemistry & Biochemistry, California State University, Long Beach, Presenting Author
Schrieffer, Matthew; Graduate, Chemistry & Biochemistry, California State University, Long Beach
Grintsevich, Elena; Faculty, Chemistry & Biochemistry, California State University, Long Beach
Abstract Actin cytoskeleton is critical for neuronal shape and function. Neuron-specific drebrin A and ubiquitous diaphanous formin-2 (mDia2), are key regulators of the actin cytoskeleton. Drebrin A is highly enriched in postsynaptic terminals of mature neurons (dendritic spines, DS) and mDia2 formin is up-regulated during dendritic spine initiation. The disappearance of drebrin from dendritic spines has been shown to be a hallmark of Alzheimer's disease, epilepsy and other complex disorders. Characterization of the interface between formin and drebrin is necessary to probe the importance of this interaction through the loss-of-binding mutant(s) in vivo. Likewise, characterization of this interface may shed light on yet unknown regulators formin. Prior work demonstrated that N-terminal sequence of drebrin (Drb1-300) contains a weak interacting site of its multi-pronged interface with mDia2. The proposed model suggested that Drb1-300 interacts with C-terminal sequence of mDia2 containing the DAD domain which involved formin’s autoinhibition through its interaction with the N-terminal DID domain. We used recombinant DID-domain to test whether Drb1-300 can relieve this autoinhibition by competing off DID-DAD interaction. Our actin assembly assay showed that the DAD domain is not involved in the Drb1-300 binding interface as the autoinhibitory complex was not outcompeted. The role of mDia2 in regulating cytoskeletal dynamics extends also to microtubules, which mDia2 is known to bind. To determine if drebrin and microtubules binding interfaces on formin overlap, we performed high-speed pelleting assay. Our results suggest non-overlapping binding interfaces for drebrin and microtubules on mDia2 formin. Next, we set out to identify high affinity mDia2 binding site in C-terminal sequence of drebrin (301-706). Mass spectrometry and in silico analysis of known formin interacting factors guided generation of an array of deletion mutants in neuronal drebrin A. We employed these mutants for pull-down assays with GST-tagged mDia2. Our findings implicate the highly conserved sequence 684-706 of drebrin as the principal site of mDia2 binding. Our investigation resulted in the determination of the drebrin sequence responsible for mDia2 binding. Our work has contributed towards designing a drebrin mutant defective in formin binding to define the biological significance of the mDia2-drebrin interaction on a cellular level.
| Poster #: 46 Campus: California State University, Long Beach Poster Category: Bioengineering Keywords: thermoresponsive carrier, drug delivery, flow cell Project Title: Peptide-based nanocarrier folding kinetics and its influence on nanocarrier uptake for drug delivery applications Author List: Sarkisian, Victoria; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author, Nagel Award Nominee
Lawrence, Katlyn; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Slowinska, Katarzyna; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract Cancer is the second leading cause of death in the United States. Next to surgery and radiation treatments, chemotherapy and combination therapies involving drugs are most successful in increasing patient survival rates. Although these therapies are effective, many of the cancer drugs exhibit systemic and non-selective toxicity to all tissues. To diminish systemic side effects, we present a simple thermo-responsive delivery system based on peptide folding mechanism. The nanocarrier, a short peptide, adopts inactive coil conformation (unfolded) at HIGH (37°C) temperatures and a helix conformation (folded) at LOW temperature. Only peptides in a helix conformation can penetrate cell membranes and deliver cargo (Fluorescein, FITC). Thus, by locally cooling the cells to 15°C (tumor model), we administer the cargo to cancer cells only, which should result in diminished side effects. While the “static” approach is very effective in delivering cargo (FITC, Paclitaxel), here we present the “kinetic” approach that involves a flow system that mimics the blood flow rate in tumor proximity. We used the Ibidi flow chamber with 80% confluent Swiss Mice Fibroblast (3T3) cell culture in an incubator with a specially designed cooling device to achieve a proper temperature gradient. Temperature is monitored with IR camera. The delivery of cargo that models the drug, FITC, is measured by analysis of images collected with Nikon Ti2 fluorescent microscope. Here we show the results were 3T3 cells were treated with solution containing peptide carrier conjugated to FITC in the flow chamber and in the static experiment. The flow chamber was exposed to a 0.3 mm/s flow rate (0.015 μL/min), similar to the rate of blood flow in capillary veins. The cell uptake was quantified, and the local temperature of the 3T3 cells was correlated with the uptake. Preliminary data indicates that with a proper temperature gradient, we can demonstrate that the kinetic rate at which the peptide achieves helix conformation is faster than the blood flow rate of capillary veins. Therefore, thermo-responsive peptide-based drug carriers are a promising candidate for use in selective drug delivery. We envision that this system could be applied in the clinical treatment of solid tumors and diminish the side effects of cancer drugs. This work was funded by the National Institutes of Health GM099594. | Poster #: 47 Campus: San José State University Poster Category: Bioengineering Keywords: bileaflet mechanical heart valves, Particle Image Velocimetry, Shear stress Project Title: PIV investigation of Bileaflet Mechanical Heart Valve Hydrodynamics Author List: Chakraborty, Sreyashi; Postdoc, Biomedical Engineering, San José State University, Presenting Author
Bellofiore, Alessandro; Faculty, Biomedical Engineering, San José State University Abstract Bileaflet mechanical heart valves (BMHVs) are the popular choices for aortic valve replacement owing to their longer durability and low cost, specially in children and low-income countries. However, they are susceptible to thromboembolism requiring lifelong anticoagulant therapy. Current research attempts to investigate the flow downstream of a BMHV that is driven by a pulsatile pressure gradient and involves dynamic fluid structure interactions. A physiological scale mock circulation loop is designed to drive flow from a super pump that produces physiological waveforms. An optically clear test-section with a St. Jude Regent 27 mm BMHV inside is installed in the mock circulation loop (MCL). A homogeneous mixture of glycerin, water and sodium thiocyanate with appropriate dynamic viscosity and refractive index is circulated as the blood analogue in the MCL. Pressure sensors on the loop measure the pressure difference across the test-section. A magnetic flowmeter measures the flow waveform upstream of the test-section. Particle image velocimetry (PIV) is used to make high-resolution measurements for pulsatile flow downstream of the valve at three different levels of vascular resistance and three different measures of compliance. Viscous shear stresses and Reynolds stresses are calculated from the spatial gradients of the Eulerian velocity field measured by the PIV. Lagrangian tracks are obtained from shear stress maps. Numerically derived blood damage index models are integrated along the Lagrangian cell trajectories to investigate the risk of platelet activation and thrombus formation at different hemodynamic conditions. The experiment is repeated with 5 commercially available BMHVs. These results will support the quantification of distinctive features of the flow that may be associated with platelet activation and coagulation, namely elevated shear stresses and areas of flow recirculation. Future tests repeated at different levels of initial clot deposition on the BMHV will be analyzed using appropriate statistical tools to correlate the extent of thrombosis on the valve surfaces with the intensity of viscous shear stresses, maximum flow velocities, and platelet damage indices. | Poster #: 48 Campus: California State University, Fresno Poster Category: Bioengineering Keywords: plasma, essential oils, wound dressings Project Title: Using continuous and pulsed 1,8-cineole plasma to modify commercially-available wound dressing materials Author List: Kayaian, Mia-Rose; Undergraduate, Chemistry and Biochemstry, California State University, Fresno, Presenting Author
Hawker, Morgan; Faculty, Chemistry and Biochemistry, California State University, Fresno Abstract Hospital-acquired infections (HAIs) are one of the leading causes of death. Specifically, HAIs are estimated to cost the United States anywhere from 28.1 to 96.8 billion dollars yearly according to Medicare data. Most HAIs begin when bacteria attach to the wound. If these wounds are left untreated, the infection site can become chronic. Existing clinical standards to treat chronic wounds include topical and oral antibiotics, but these can become problematic because of possible antibiotic resistance. Designing a wound dressing to actively kill bacteria on contact would be optimal for chronic wound treatment. Plasma-enhanced chemical vapor deposition (PECVD) using 1,8-cineole as the plasma feed gas has been shown to actively kill bacteria on glass slide surfaces. Yet, 1,8-cineole-based PECVD strategies have not been extended to wound dressing materials.
The goal of the present study is to use 1,8-cineole PECVD to modify commercially-available wound dressing materials (hydrofibers and hydropolymers). Our strategy was to deposit a film with antibacterial properties using plasma treatment. By adjusting plasma parameters, it is possible to control the coating chemistry of the chosen materials. Importantly, no attempt has been made to compare pulsing vs. continuous-wave deposition. This represents a promising strategy for the current work because pulsing power enabled the plasma precursor’s functional groups to be maintained to a greater degree (when compared to continuous power) for other PECVD precursors. We hypothesized that pulsing would enable the functional groups to be maintained upon PECVD since the functional groups give 1,8-cineole its antibacterial properties. This study utilized optimized plasma parameters (P=20 W, treatment time = 20 min) for continuous and pulsed depositions. The pulsed depositions utilized were 10%, 25%, and 50% duty cycles. X-ray photoelectron spectroscopy was used to determine the elemental composition of the plasma-treated and control surfaces, and water contact angle goniometry was used to evaluate changes in surface wettability of the materials before and after plasma treatment. Zone of inhibition testing was performed to evaluate antibacterial properties against gram-positive and gram-negative bacteria. Overall, this work represents progress toward directly targeting chronic wound infections. | Poster #: 49 Campus: California State University, Long Beach Poster Category: Bioengineering Keywords: droplets, microfluidics Project Title: Millifluidic Droplet Generation Author List: Thai , Chung ; Undergraduate, Biomedical Engineering, California State University, Long Beach
Ametepe, Brenda A.A.B. ; Undergraduate, Biomedical Engineering, Presenting Author
Simon, Melinda ; Faculty, Biomedical Engineering, San José State University
Ayala, Perla; Faculty, California State University, Long Beach
Ahrar, Siavash ; Faculty, Biomedical Engineering, California State University, Long Beach
Abstract Much attention has been paid to the systematic development of microfluidic droplet generators that produce droplets in the pL-nL volume range. In turn, microfluidic droplet generators have enabled numerous exciting research applications (e.g., high throughput screening, sequencing, diagnostics) and led to successfully commercialized systems. However, a well-controlled generation of uniform mm-scale droplets that provide higher working volumes could enable new technologies for exciting applications, particularly in biomanufacturing and tissue engineering. Towards this aim, we developed a rapid and inexpensive prototyping approach using laser-cutting and tape sealing. We used the prototyping to establish millifluidic droplet generators based on a hydrodynamic focusing architecture. We also demonstrated that similar to microfluidic systems, droplet size and generation frequency could be adjusted by modulating the relative flow rate ratios. Our dimensionless-number analysis (i.e., the Capillary number) suggested that the presented millifluidic systems operate in the "squeezing" regime of droplet generation. This analysis also suggested that, despite larger system dimensions, the interfacial tension forces remain dominant over gravity. Next, we demonstrated alternative droplet generator designs (e.g., droplet traps) via frugal prototyping. Droplet generators with
“merging-zone” and droplets as large as 20.37 +/- 1.2 mm^2 were demonstrated. Finally, we used millifluidics to generate mm-scale hydrogel beads (photopolymerized PEG) and Matrigel droplets to demonstrate the millifluidics utility in future applications. PEG and Matrigel were selected as
representative classes of materials commonly used in biomaterials and tissue engineering applications. The presented prototyping approach, designs, and applications provide a framework for millifluidic droplet generation and further extend the application space for droplets.
| Poster #: 50 Campus: San José State University Poster Category: Bioengineering Keywords: Phosphate metabolism, Rare earth elements, Methylobacterium extorquens Project Title: Investigation of phosphate metabolism in Methylobacterium extorquens for enhanced bio-recycling of rare earth metals from electronic waste Author List: Kaur, Harpreet; Undergraduate, Biological Sciences, San José State University, Presenting Author
Numan, Thasel; Undergraduate, Biological Sciences, San José State University, Presenting Author
Tizon, Colan; Undergraduate, Biological Sciences, San José State University
Enrile, Meagan; Undergraduate, Biological Sciences, San José State University
Trinidad, Caitlyn; Undergraduate, Biological Sciences, San José State University
Nguyen, Tiffany ; Undergraduate, Biological Sciences, San José State University
Rasouli, Sajede; Graduate, Biological Sciences, San José State University
Shao, Eric; Undergraduate, Biological Sciences, San José State University
Skovran, Elizabeth; Faculty, Biological Sciences, San José State University
Abstract Rare earth elements (REEs) are critical components of electronic devices and green energy technologies. However, mining for REEs is environmentally destructive and toxic to humans. We have shown that the methylotrophic bacterium, Methylobacterium extorquens, can acquire insoluble REEs from electronic waste. These REEs are then used as cofactors for methanol oxidation enzymes or stored in polyphosphate granules, which can be extracted for REE recovery. For this bio-recycling platform to be commercially viable, REE/phosphate (Pi) uptake and storage must be increased through genetic engineering. However, Pi metabolism has not been studied in this organism. To investigate the connection between REEs and Pi, we first established conditions of Pi excess and starvation in wild type and methanol oxidation mutant strains. Surprisingly, growth analysis of methanol oxidation mutants revealed the existence of a novel methanol oxidation system that operates during Pi starvation. Using a modified Albert’s stain, we visualized REE-Pi granules and show that these granules are depleted during Pi starvation. We used comparative genomics to identify pstSCAB as a potential Pi uptake system. Using transcriptional reporter fusion assays, we show that expression from the pstSCAB promoter is regulated by exogenous REEs in addition to Pi. These data suggest a homeostatic connection that coordinates methanol oxidation, Pi and REE levels. To engineer increased Pi uptake for our bio-recycling platform, we attempted to construct mutations in the phoU Pi uptake regulatory gene. Repeated attempts failed suggesting phoU may be essential. Future studies will use transposon mutagenesis and transcriptomics to identify the novel methanol oxidation system. To increase Pi uptake and storage without having to mutate phoU, we will test if M. extorquens can hyperaccumulate Pi after alternating between carbon and Pi replete and deplete conditions, which induces Pi accumulation in bacteria used in wastewater treatment. This work lays the foundation to support genetic engineering efforts to enhance REE recovery yields from electronic waste and was funded by a Department of Energy ARPA-E grant and two San Jose State University Student Research Fellowships. | Poster #: 51 Campus: California State University, Long Beach Poster Category: Bioengineering Keywords: Cardiac Fibrosis, Fibroblasts, Collagen VI Project Title: Development of 3D Fibrosis Models-Effect of Critical ECM Components on Fibroblast Function Author List: Zavala, Jane; Undergraduate, Biomedical Engineering, California State University, Long Beach, Presenting Author
Tran, Khanh; Undergraduate, Biomedical Engineering, California State University, Long Beach, Presenting Author
Keu, Kim; Graduate, Biomedical Engineering, California State University, Long Beach
Ayala, Perla; Faculty, Biomedical Engineering, California State University, Long Beach Abstract
After a myocardial infarction, the adult mammalian heart has a limited ability to regenerate, and the considerable loss of cardiac cells induces the formation of scar tissue, known as cardiac fibrosis. It is characterized by an excessive extracellular matrix (ECM) deposition which can result in significant tissue stiffening, cardiac tissue remodeling, and heart dysfunction, potentially leading to cardiac failure and death. There are currently no viable therapies to reverse chronic fibrosis. Using unique 3D fibrosis models, we investigate ways of modulating the cardiac microenvironment on fibrosis progression. The hypothesis is that the introduction of defined matrix proteins can either promote or diminish the development of fibrotic conditions in the constructed 3D fibrosis model. CCN1 (also known as CYR61; cysteine-rich protein 61) and Collagen type VI (Col VI) impact fibrotic depositions, making them appealing candidates for inclusion in 3D engineered fibrosis models. The present model is made up of a 2.5 mg/mL starting concentration of 3D collagen type I hydrogel. Fibroblasts were cultured within these 3D models with or without CCN1 or Col VI for five days. To analyze cell morphology, F-actin was stained with an Alexa-phalloidin conjugate, myofibroblasts were immunostained with an alpha-smooth muscle actin (α-SMA) antibody, and nuclei were stained with DAPI. We analyzed changes in fibroblast numbers and α-SMA expression using fluorescence imaging and Image J. Quantitative polymerase chain reaction (qPCR) was also performed for the following targets: GAPDH, α-SMA, Cyclin D1. Preliminary data suggest that fibroblasts seeded in models with CCN1 show decreased proliferation. The expression of α-SMA in fibroblasts was increased by collagen type VI inclusion, despite the fact that there was no discernible lower fibroblast proliferation. In vitro 3D models of post-MI remodeling showed that the presence of collagen type VI, which is known to be a critical ECM component, might promote myofibroblast differentiation. Results from this study could help improve methods focused on repairing cardiac tissue and heart functionality.
| Poster #: 52 Campus: San José State University Poster Category: Bioengineering Keywords: Mechanobiology, Coagulation, Blood Project Title: Role of factor XIII on fibrin clot structure and mechanics Author List: Awan, Myra; Graduate, Chemical and Materials Engineering, San José State University, Presenting Author
Cheng, Terrence; Staff, Chemical and Materials Engineering, San José State University
Walvekar, Ankita; Graduate, Chemical and Materials Engineering, San José State University
Ramasubramanian, Anand; Faculty, Chemical and Materials Engineering, San José State University Abstract Fibrin is the key structural and functional component of blood clot. Fibrin is a highly extensible, viscoelastic, strain-stiffening material. Fibrin fibers are formed by the polymerization of monomeric fibrinogen, a process catalyzed by thrombin in the presence of calcium. The 3D fibrin network is formed by the branching of fibers, and the network is strengthened by covalent linkages within and between fibers. This covalent crosslinking is mediated by the transglutaminase FXIIIA. FXIIIA is present in both plasma, and is also expressed on the surface of activated platelets and other cells of bone marrow origin. In contrast to plasma FXIIIA, the role and importance of FXIIIA expressed on the cell surface is poorly understood. In this work, we have investigated the role of surface-bound FXIIIA on fibrin crosslinking, structure, and mechanics using FXIIIA conjugated to carboxylated microspheres in FXIII-free plasma. We quantified spatial variations in fibrin network structure using fluorescence and electron microscopy, and image analysis; and evaluated the mechanical properties of the bead-encapsulated clots using rheometry. We observed that surface-bound FXIIIA crosslinked fibrin on bead surface resulting in increase in local fibrin density, and protects fibrin from enzymatic lysis. We also observed that surface-bound FXIIIA increases the elastic modulus of the clots. Together, our results show that even in the absence of plasma FXIII, cellular FXIII increased fibrin network density locally; and suggest that cellular FXIII may play an important role in hemostasis and wound healing. | Poster #: 53 Campus: California State University, Fresno Poster Category: Bioengineering Keywords: Gold nanoparticles, Protein cages, Self-assembly Project Title: Encapsulation of gold nanoparticles into protein cages through genetically modified viral proteins Author List: Clark, Amber; Graduate, Chemistry and Biochemistry, California State University, Fresno, Presenting Author
Fukazawa, Risako; Staff, Chemistry and Biochemistry, California State University, Fresno
Gutierrez, John; Undergraduate, Chemistry and Biochemistry, California State University, Fresno, Presenting Author
Echeveria, Dustin; Staff, Chemistry and Biochemistry, California State University, Fresno
Espinoza, Randy; University of California, Merced
Nguyen, Son; University of California, Merced
Uchida, Masaki; Faculty, Chemistry and Biochemistry, California State University, Fresno
Abstract Protein cages are small, hollow, spherical, protein structures such as virus-like particles (VLPs), ferritins, and encapsulins. Their ability to self-assemble into highly organized cage-like structures with an inner cavity make them an attractive platform for encapsulating guest cargos including inorganic nanoparticles. Combining a biological protein cage with inorganic material allows the container-cargo unit to take on unique chemical and physical properties characteristic of both biological and abiotic materials. In this study, we utilize VLPs derived from bacteriophage P22 as a protein cage platform to encapsulate gold nanoparticles. P22 VLP is constituted with 420 copies of coat protein (CP) and approximately 100 – 300 copies of scaffolding protein (SP). Interestingly, P22 VLPs can be formed by mixing individual CP and SP monomers in vitro as well as recombinantly co-expressing these proteins in E. coli. We hypothesized that if SPs can associate with gold nanoparticles, CPs will self-assemble around the gold nanoparticles and encapsulate them inside of the VLPs. Because wild-type SP does not naturally form specific interactions with gold nanoparticles, we have genetically introduced a peptide motif, which shows specific affinity with gold nanoparticles, at the N-terminus of the SP. We have shown that the engineered SP maintains the capability to mediate assembly of CP into VLPs in the same manner as wild-type SP through in vitro assembly in the absence of cargo. The hydrodynamic radii (Rh) of the VLPs post-in vitro assembly were confirmed using dynamic light scattering techniques. Measured Rh of the VLPs with the engineered SPs as well as VLPs with the wild-type SPs were around 30 nm which is consistent with Rh of P22 virus capsid. We are currently investigating the encapsulation of gold nanoparticles inside P22 VLPs by using the engineered SP and wild-type CP. Successful encapsulation of gold nanoparticles will be confirmed using transmission electron microscopy. It is anticipated that the findings will highlight the particular specificity between the SPs and cargo required for efficient encapsulation of gold nanoparticles.
Acknowledgments: We thank CSUPERB Faculty-Graduate Student Research Collaboration Program, CSU Fresno College of Science and Mathematics FSSRA, and the National Science Foundation grants CMMI-1922883 for support of this work.
| Poster #: 54 Campus: California State University, Fresno Poster Category: Bioengineering Keywords: Linker mediated protein assembly, P22 virus-like particle, Computational modeling Project Title: Evaluating Assembly of P22 Virus-Like Particle (VLP) Mediated by Dec-based Linker Proteins Author List: Medina, Paulina ; Undergraduate, Biology, California State University, Fresno, Presenting Author
Fukazawa, Risako; Staff, Chemistry and Biochemistry, California State University, Fresno
Lopez, Amalia; Undergraduate, Chemistry and Biochemistry, California State University, Fresno, Presenting Author
Terashi, Genki; Purdue University
Kihara, Daisuke; Purdue University
Uchida, Masaki; Faculty, Chemistry and Biochemistry, California State University, Fresno Abstract Virus capsids and protein cages are used as building blocks with the ability to form higher order three-dimensional array materials. Higher order assembly serves in biomaterial applications such as encapsulation of guest molecules and catalysis. In our laboratory, virus-like particles (VLPs) derived from bacteriophage P22 have been studied as a building block to form higher order assembly. One mechanism by which such a higher order structure can be achieved is through the utilization of decoration (Dec) protein. Dec serves as a linker to assist the P22 VLPs in higher order assembly, such as dimer structure, by binding with its exterior surface. P22 has two forms: procapsid (PC) form and expanded (Ex) form. It has been successfully demonstrated that Dec protein can be used to facilitate assembly of P22 in its Ex form, however the original Dec protein does not have the ability to bind to the PC form of P22. For the purpose of materials development, it is advantageous to mediate assembly of the PC form of P22. This research therefore aims to engineer Dec-based linker proteins to investigate the assembly of P22 in its PC form.
Computational modeling has been performed by our research team in Kihara Lab at Purdue University to evaluate protein structures which have affinity to the PC form of P22. The result suggests that a helix-turn-helix (HTH) bundle has some affinity to attach to P22 PC. LZerD Protein-Protein Docking is used to estimate the binding affinity of P22 PC with three types of HTH bundles with P22 PC: gp41 core, derived from HIV protein; and 2L6HC3_13 and 5L6HC3_1, produced from de novo protein design. The protein docking approaches indicate that gp41 core has the highest binding prospect to P22 PC. Next, we experimentally designed Dec based linker proteins formulated on these computational predictions. We have constructed plasmids for expressing the three fusion proteins, and furthermore transformed the plasmids to E. coli. We are currently evaluating expression and the capability of these proteins to mediate higher order assembly of P22 PC. We expect that integration of computational modeling and experimental verification will be a powerful approach to design protein linkers for controlled construction of protein array materials.
Acknowledgments: We thank the National Science Foundation (NSF) Grants CMMI-1922883, the National Institute of Health (NIH) Grant R25GM131956, and CSU-Fresno CSM FSSRA for support of this work.
| Poster #: 55 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Bioengineering Keywords: Angiogenesis, Preclinical Drug Testing, Tissue Engineering Project Title: The Effect of SN-38 and Oxaliplatin on an Injectable 3-Dimensional Endothelial Network Model Author List: Stuehr, Eric; Graduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Adams, Ryan; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Heylman, Christopher; Faculty, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Abstract The pharmaceutical industry spends 2.5 billion dollars annually on preclinical testing for chemotherapies. Current disease models for preclinical testing include monolayer cultures and animal models. However, when a drug moves to clinical trials, it is often discovered that these models were inadequate and failed to reproduce the physiological in-vivo response observed in humans, causing years of time and money to be wasted. Therefore, there is a need for an improved model to be used for testing the effects of chemotherapeutic agents. In vivo, chemotherapies are delivered to tissues via vascular networks. Therefore, development of a vascularized tumor microenvironment can offer a better model of in vivo responses to chemotherapies during preclinical drug testing. It is also important to characterize vascular network response to chemotherapies alone to understand if there are any off-target effects. We have developed an injectable 3-dimensional vasculature model. The model, which consists of a coculture of human umbilical vascular endothelial cells (HUVECs) and human dermal fibroblasts (HDF), has successfully established endothelial networks within in vitro cultures. Optimization of formation and sustention of endothelial networks was performed, as well as testing chemotherapeutic agents (SN-38 and Oxaliplatin) for off-target effects on endothelial network formation. The key parameters that were optimized were cell densities of HDFs and HUVECs, Matrigel extracellular matrix density, and loading conditions in the microfluidic device. SN-38 and Oxaliplatin were shown to have minimal effects on endothelial network formation at clinical dosing for colorectal cancer treatment. Future work will be conducted to further optimize endothelial networks using hypoxic conditions and Vascular Endothelial Growth Factor (VEGF) gradients to better mimic the in vivo niche. Current and future work also aims to integrate optimized endothelial networks with colorectal cancer cells via an injectable 3D matrix within a microfluidic device for the purpose of establishing an accurate model of a vascularized tumor environment for preclinical drug testing. | Poster #: 56 Campus: San José State University Poster Category: Clinical Keywords: metaboreflex, exercise pressor reflex, parasympathetic nervous system activity Project Title: Does the Vagal Tone Influence Metaboreceptor-mediated Pressor Responses in Humans? Author List: Wang, Stephanie; Undergraduate, Kinesiology, San José State University, Presenting Author
Pizano, Matthew; Undergraduate, Kinesiology, San José State University
Carlota, Gabriel; Undergraduate, Kinesiology, San José State University
Bui, Allison; Graduate, Kinesiology, San José State University
Jensen, Areum ; Faculty, Kinesiology, San José State University
Abstract Exercise is associated with parasympathetic (i.e., vagal) withdrawal and sympathetic activation resulting in an increase in heart rate (HR), stroke volume (SV), and vascular resistance; thus increase in blood pressure (BP). Activation of exercise pressor reflex via mechano (group III) and metabo (group IV) receptors from contracting skeletal muscles contributes to increase in sympathetic outflow which modulates BP to meet the higher metabolic demands for targeted exercise. Post-exercise cardiodeceleration is due to vagal reactivation which appears to be related to resting vagal tone. Thus, we thought to investigate the role of vagal reactivation after exercise when the sympathetic nervous system is still highly activated by metaboreceptors. A total of 17 young adults participated in the study. HR from ECG, beat to beat arterial BP from Finapres, and SV from Modelflow, were continuously measured during baseline, 2 minutes of handgrip exercise at 35% of maximal contraction, 2 minutes of occlusion on the exercising arm (post exercise ischemia; PEI), and 3 minutes of recovery. Time and frequency domains of HR variability indexes were analyzed from 5 minutes of resting baseline segment, and used to assess vagal tone. High frequency (HF) domain was used to represent higher HR variability. Mean BP had a similar increase from rest to exercise (Δ12mmHg) and remained high from exercise to PEI (Δ1-2mmHg) regardless of groups (high HF vs. low HF or males vs. females). Both high and low HF groups had similar increases in HR (Δ7.8±1.5 in High HF vs Δ10.1±1.3 in low HF, bpm) from rest to exercise and decrease in HR from exercise to PEI (Δ3.8±1.9 in High HF vs Δ3.1±0.8 in low HF, bpm). However, males exhibited greater increases in HR from rest to exercise (Δ10.1±1.2 in males vs Δ6.9±2.2 in females, bpm) and had significant decrease in HR from exercise to PEI compared to females (Δ7.2±0.9 in males vs Δ2.2±1.6 in females, bpm). In conclusion, high or low vagal tone may not affect the strength of post-exercise vagal reactivation while the sympathetic nervous system is still activated by metaboreceptors and BP is still augmented. Males and females may have different mechanisms to regulate BP by exercise pressor reflex. Males may rely less on cardiac contributions to exercise pressor response even though vagal reactivation was greater.
This work is supported by National Institute of Health, National Institute of General Medical Sciences Grant (SC2GM144165 to A.K. Jensen)
| Poster #: 57 Campus: California State University, Long Beach Poster Category: Keywords: Adolescence, Addiction, Animal Model Project Title: Activation of 5-HT1B Receptors Attenuates the Acquisition of Nicotine Reward in Adolescent Male Rats. Author List: Garcia, Ana; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Ponce, Hannah; Graduate, Psychology, California State University, Long Beach, Presenting Author
Gonzalez-Gutierrez, Tiffany; Graduate, Psychology, California State University, Long Beach
Zavala, Arturo; Faculty, Psychology, California State University, Long Beach Abstract Activation of serotonin (5-HT)1B receptors decreases the rewarding and reinforcing effects of stimulant drugs, such as cocaine and methamphetamine. In the present study, we examined the hypothesis that administration of CP 94,253, a 5-HT1B receptor agonist, would reduce nicotine preference in adolescent male rats using a 10-day Conditioned Place Preference (CPP) procedure, a well-established animal model of drug reward. On postnatal day (PD) 28, baseline preference for a two-sided apparatus was assessed during a 15 min session. In two-day cycles, rats received an injection of CP 94,253 (0 or 5.6 mg/kg) 15 min before the administration of nicotine (0, 0.2, or 0.6 mg/kg) on one day and saline administration on the other day before being confined to one side of the two-chamber apparatus for 15 min. This two-day cycle was repeated over the next 6 days. On day 10, the preference for the nicotine-paired chamber was assessed for 15 min. Rats exhibited nicotine-induced CPP when conditioned with either 0.2 or 0.6 mg/kg of nicotine. Administration of CP 94,253 (5.6 mg/kg) before nicotine (0.2 or 0.6 mg/kg) resulted in a decreased preference for the nicotine-paired compartment. The present findings demonstrate that activation of 5-HT1B receptors with CP 94,253 attenuated the acquisition of nicotine-induced CPP in male adolescent rats. Overall, these findings further add to a growing body of literature that points to the 5-HT1B receptor as a pharmacological target for treating psychostimulant addiction. | Poster #: 58 Campus: California State University, Long Beach Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Vitamin D, Molecular Dynamics, Computational Chemistry Project Title: Membrane enhances vitamin D production by stabilization of helical previtamin D conformers Author List: Plazola, Matthew; Undergraduate, Matthew.Plazola@student.csulb.edu, California State University, Long Beach, Presenting Author
Smith, Adam; Graduate, Chemistry and Biochemistry, California State University, Long Beach
Tapavicza, Enrico; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract The photochemical reactivity of vitamin D derivatives depends on excitation wavelength and chemical environment. This dependency is mainly governed by the conformational flexibility of previtamin D (PreD), the central compound in vitamin D photoequilibrium. To assess the influence of the phospholipid bilayer on natural vitamin D photosynthesis, we studied conformational equilibrium of previtamin D in dipalmitoylphosphatidylcholine (DPPC) phospholipid bilayers using classical molecular dynamics simulations. An accurate description of the torsional potential energy of previtamin D requires a balanced description of steric repulsion and π-orbital conjugation of its central hexatriene unit. To achieve this, we applied a correction map (CMAP) based on density functional theory to the CHARMM generalized force field (CGenFF). To sample the thermodynamic limit of the distribution of conformers, we applied the enhanced sampling methods replica exchange molecular dynamics and the adaptive biasing force sampling technique. Our simulations show that the DPPC bilayer leads to a stabilization of the helical g+Zg+ and g-Zg- conformers. Since the sigmatropic [1,7]-hydrogen transfer forming vitamin D is only possible in helical conformers, this explains the experimentally found 10-fold increase of thermal vitamin D formation rate in membranes compared to isotropic solutions. In addition, we show that photoinduced ring-opening of 7-dehydrocholesterol leads to a reduction of the order parameter of the membrane. The order parameter of the DPPC/previtamin D system, however, is still larger than for a pure DPPC membrane.
| Poster #: 59 Campus: California State University, Long Beach Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: protein docking , computational biology Project Title: FMOC Leucine Amino Acid Inhibitors Exemplify Strong Noncovalent Interactions in Complex with Butyrylcholinesterase Enzyme Author List: Aceituno, Karel; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Mao , Jia; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach
Schwans , Jason ; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Sorin, Eric ; Faculty, Chemistry and Biochemistry, California State University, Long Beach Abstract The formation of amyloid beta plaques is a prominent indicator of the progression of Alzheimer’s Disease (AD); this symptom is also associated with overactivity of the butyrylcholinesterase (BChE) enzyme, which hydrolyzes the neurotransmitter acetylcholine, making it a plausible target for the treatment of this neurodegenerative disease. Here, computational methods were used to probe the interactions between the enzyme and inhibitor candidates featuring 9-fluorenylmethyloxycarbonyl (Fmoc). Using Molsoft ICM-Pro software, 10000 docking trials of 12 Fmoc inhibitors were performed, and trials resulting in the most optimal docking scores were examined. The best candidate was Fmoc-Norleucine, consistent with experimental IC50 values. Analysis of the best-scoring structures show that the length of the aliphatic R group in inhibitors was most influential on resulting docking scores, as the four inhibitors with the largest side chains (L-Norleucine, D-Norleucine, L-Homoleucine, and D-Homoleucine) had the most favorable docking scores. From visual analyses of the enzyme in complex with these inhibitors, their larger aliphatic groups allowed for more significant 𝜋-stacking between Fmoc and peripheral aromatic site residues of BChE, as well as orientation of the terminal carboxylate of inhibitors farther into the enzyme active site near the oxyanion hole and catalytic triad, both of which are hydrogen-rich and thus favor hydrogen bonding interactions. Identification of trends observed in experimental and in silico studies provides a better foundation for future design of selective amino acid-based BChE inhibitors.
This project is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R25GM071638. | Poster #: 60 Campus: California State University Channel Islands Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Nucleoside Analogues, Pancreatic Cancer, Molecular docking Project Title: Molecular docking, ADME, and PASS predictions of novel polyethylene glycol-amino modified nucleosides as potential pancreatic cancer therapeutics Author List: Drobshoff, Aleksya; Undergraduate, Chemistry, California State University Channel Islands, Presenting Author
Khosroabadi, Delyar; Undergraduate, Chemistry, California State University Channel Islands, Presenting Author
Awad, Ahmed; Faculty, Chemistry, California State University Channel Islands
Abstract Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer with a high mortality rate due to a lack of early detection techniques leading to diagnosis at a terminal stage and limiting treatment options. The nucleoside analogue Gemcitabine, while the most effective chemotherapeutic for PDAC, faces drug resistance that hinders its therapeutic potential. In vivo activation of this prodrug involves phosphorylation by deoxycytidine kinase to the diphosphate form that binds within the catalytic site of ribonucleotide reductase (RNR), the key target for PDAC treatment. We propose nucleoside analogues that feature a polyethylene glycol amino (PEGA) modification at the C2’ position of the ribose sugar. This modification replaces the two fluorine atoms in the corresponding gemcitabine structure and hence reduces the hydrophilic nature of this drug. The PEGA group is also hypothesized to serve as a chelating agent for both zinc and iron ions required for RNR activity. Molecular docking using the internal coordinate mechanics algorithm (ICM), SWISS Absorption, Distribution, Metabolism and Excretion (SWISSADME), and prediction of activity spectra for substance (PASS), utilizing way2drug online application were performed on the proposed molecules. The edock scores, the root mean square deviation (RMSD), and the H-bonding formation were analyzed. Edock scores showed a higher binding affinity than the reference drug gemcitabine, with an average value of -48.52 KJ/mol and an average RMSD value of 2.69. The PEGA moiety showed a strong binding affinity to a known substrate binding residues in the catalytic sites such as the amino acid Asn211, a key component in the catalytic processes. The pharmacokinetics and toxicity were assessed through SWISSADME which demonstrated excellent solubility scores of logS -0.37 and showed non-toxic inhibition interactions to CYP isoenzymes. Through PASS prediction, the biological activity of the compound showed high potential as an RNR inhibitor with a probability of activity (Pa) to probability of inactivity (Pi) of 0.174 to 0.011. In conclusion, the proposed analogues demonstrate improved target inhibitions making them strong candidates for further investigation via in vivo and in vitro studies. | Poster #: 61 Campus: California State University, Fresno Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: signal averaging, photoacoustic spectroscopy, blood glucose monitoring Project Title: Photoacoustic Spectroscopy for Blood Glucose Monitoring with Light Emitting Diodes utilizing Signal Averaging Techniques Author List: Srinivas, Rithika Bollure; Graduate, Electrical and Computer Engineering, California State University, Fresno, Presenting Author
Shaikh, Faheem; Graduate, Electrical and Computer Engineering, California State University, Fresno
Laha, Soumyasanta; Faculty, Electrical and Computer Engineering, California State University, Fresno
Abstract The project proposes to design a non-invasive continuous glucose monitoring system following the technique of photoacoustic spectroscopy using the light emitting diodes (LEDs). Until now, lasers have been used to excite the glucose solutions to know its concentration using the technique of photoacoustic spectroscopy. The use of lasers has multiple safety concerns and hence not recommended for in-vivo blood glucose monitoring applications. To make photo-acoustic spectroscopy feasible to in-vivo blood glucose monitoring in humans, the LEDs are needed to replace lasers considering the safety issues. However, the signal to noise ratio (SNR) obtained using LEDs is not of satisfactory level in comparable to that of using lasers. To achieve comparable SNR to that of lasers with LEDs, in this project an advanced signal averaging technique has been developed. The algorithm is then implemented in a power efficient micro-controller to transform the weaker light signal of LEDs into a high SNR processed signal for practical in-vivo application of photo-acoustic spectroscopy in humans. To the best knowledge of the authors, this is the first work to report photoacoustic spectroscopy using LEDs implementing signal averaging techniques for blood glucose monitoring. | Poster #: 62 Campus: San Francisco State University Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Manduca sexta, spike sorting, neuroscience Project Title: Characterizing the neural circuitry mediating the defensive strike response after nociceptive sensitization in an insect model Author List: Valtierra, Christian; Graduate, Biology, San Francisco State University, Presenting Author
Tabuena, Dennis; Graduate, Biology, San Francisco State University
Fuse, Megumi; Faculty, Biology, San Francisco State University
Abstract Nociceptive sensitization is a heightened state of arousal resulting from a decrease in the sensory threshold required to engage in defensive behaviors following exposure to an intensely noxious or harmful stimulus, noted as nociception. The hornworm, Manduca sexta, displays rapid defensive “striking” behavior that accurately targets the cite of noxious stimulation of the abdominal body wall. Nociceptive sensitization of the defensive strike response has been induced behaviorally and recapitulated in vitro through extracellular electrophysiology, where it thought to be encoded by an increase in the firing frequency occurring within the central nervous system, rather than changes in peripheral nociceptor signaling. The current model proposes that central neural alterations are responsible for the neural correlate of nociceptive sensitization, rather than changes in spiking activity of the primary sensory neurons. However, this may be inaccurate, as the model is based on the comparison of threshold crossings of purported multiunit activity rather than of single cell activity of known populations of sensory and/or higher order neurons. Therefore, it is worth re-examining the possibility of presynaptic plasticity. As a first step towards exploring this, I propose leveraging available extracellular electrophysiology data to re-analyze a prior sensitization and habituation dataset. I will perform spike sorting and spike train analyses to tease apart the signals originating from the peripheral sensory neurons to obtain input/output relationships between the identified sensory units and central spiking during stimulation. Distinguishing the nociceptor and tracking its stimulus dependent firing is expected to answer the question of peripheral vs central alterations.Preliminary results are in line with the established neuroanatomy: spike sorting of the dorsal nerve data for samples analyzed (n = 10) has consistently recovered the signals of 4 putative neurons in both injured and non-injured CNS preparations. Future directions include expanding the analysis pipeline by including within sample spike sorting quality metrics and using machine learning to improve waveform matching between samples. | Poster #: 63 Campus: San José State University Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Neural Networks/Transformers, Natural Language Processing, High-throughput Sequencing Project Title: SRASearch - an enriched NCBI SRA search with a transformer-based neural network Author List: Kapoor, Aadit; Graduate, Department of Computer & Software Engineering, San José State University, Presenting Author
Chopra, Aarohi; Graduate, Department of Computer Science, San José State University
Lee, Wendy; Faculty, Department of Computer Science, San José State University
Abstract Biomedical text understanding and retrieval is an active field of research. Advances in genome sequencing technologies have led to an exponential increase in genome sequence data deposited in the public repository, such as the NCBI Sequence Read Archive (SRA). Despite having a vast amount of publicly available data for researchers to share and analyze, the existing NCBI SRA search capability is limited since it lacks the inclusion of semantics in its search functionality. Recent advances in Natural Language Processing (NLP) and Information Retrieval (IR) have allowed us to build search systems capable of understanding search terms' semantics. The influx of publicly available data and NLP advances such as Bidirectional Encoder Representations from Transformers (BERT) has allowed us to fine-tune our model on applicable downstream tasks such as text classification and Named Entity Recognition (NER). Here we present a search tool, SRASearch, that utilizes NLP techniques to improve search functionality from the NCBI SRA database.
SRASearch comprises contextual understanding. We utilize a popular concept in NLP known as embeddings to understand biomedical entities. Word embeddings are language representation models that use a neural network to represent words as low-dimensional vectors. Pre-trained word embeddings are fine-tuned by training them on a given choice of corpus and adding an output layer. We utilize open-source technologies to build this search platform that can categorize and search data in multiple tables, reducing the "out of search" terms. We form our hypothesis with experiments ranging from using baseline word representation methods such as one-hot encoding to more advanced techniques, such as word2vec and BERT. We employ different pre-processing steps, such as removing stop words and exploiting statistical knowledge about the words to make our data more informative. Using a combination of pre-processed SRA tables and a transformer-based model approach, we measure several manual heuristics, such as disease-gene similarity and biomedical sentence similarity. The search outputs the top five nearest neighbors with the highest cosine similarity to the search term and the index vector.
Our research shows how existing NLP and IR models can help redefine and significantly improve SRA search, providing greater flexibility to researchers and thus reducing time, complexity, and search errors. | Poster #: 64 Campus: California State University, Los Angeles Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: BCI, surface EEG, forward model Project Title: An engineered system to validate a computational forward model of surface electroencephalograms encoding movement behavior Author List: Sercel, Gregory; Undergraduate, Electrical Engineering, California State University, Los Angeles, Presenting Author
Ao, Stellina; Undergraduate, Electrical Engineering, California State University, Los Angeles, Presenting Author
Won, Deborah ; Faculty, Electrical Engineering, California State University, Los Angeles
Abstract Brain-computer interface (BCI) systems traditionally acquire neural signals via invasive electroencephalography (EEG) methods. Invasive EEG techniques pose a high risk to patients and require complex implantable hardware. In contrast, non-invasive surface EEG (sEEG) can be acquired from electrodes placed on the scalp with a significantly lower risk and lower cost; however, the signal resolution is also much lower than invasively acquired EEG. Our specific aim in the present work is to design and develop the experimentation system which will provide the data necessary to validate our computational model.
This work focuses on developing the experimental system that will enable us to model the gripping forces encoded in sEEG. Our experimental system consists of sEEG acquisition, a virtual instrument to conduct the behavioral tasks, and a force feedback circuit to acquire the behavioral data that will eventually be encoded by our model to predict the source sEEG.
sEEG is acquired with the MP160 EEG amplifier hardware (Biopac Systems), via the AcqKnowledge software. The following 5 channels of sEEG are acquired at a sampling rate of 200Hz with a gain of 1500 and two-pole bandpass filtering at cutoffs of 1 Hz and 200 Hz: Fpz, F1, F3, Oz, and Cz. The horizontal electrooculogram (EOG) channel is also acquired in order to detect and remove eyeblink artifact. A virtual instrument (or VI) was designed and coded in LabView (National Instruments) for two main purposes: 1) to guide the subject through the force gripping experiment; and 2) to send external triggers to synchronize acquisition of the subject’s sEEG from AcqKnowledge and the resulting hand gripping force from the subject via the LabView VI. The VI displays a circle whose diameter increases with the force the subject applies to a ball they are gripping. The subject is requested to apply force to the ball in their hand until the circle’s diameter matches the target circle’s diameter. The triggers are sent via serial port to AckKnowledge which as TTL pulses recorded on a separate channel. The gripping force of the subject is measured by an electrical circuit which employs an array of force sensing resistors in an array of voltage dividers, one for each finger which are placed on a foam stress ball.
This system enables us to create a BCI that has the same quality of predictions as a conventional invasive BCI but is significantly safer for the user.
| Poster #: 65 Campus: California State University, Long Beach Poster Category: Diagnostics/Imaging/Analytical Keywords: fluorescence, metal-organic framework Project Title: Activation of Solid-Phase Luminescence by Fluorescein Encapsulation within Zeolitic Imidazolate Framework Author List: Yu, Raymond; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Tian, Fangyuan; Faculty, Chemistry and Biochemistry, California State University, Long Beach Abstract Fluorescein is an important fluorescent probe with a broad range of applications. Despite its impressive breadth, they are limited to solution phase applications because fluorescein is not emissive in the solid phase due to aggregation induced quenching. In this work, fluorescein’s utility is extended to solid phase application by encapsulation into ZIF-8. The hybrid metal-organic framework was easily synthesized in one-pot and retains characteristic stability, crystallinity, porosity, and high surface area characteristic of ZIF-8 with emergent optical properties not observed in its constituents. An application for the detection of explosive molecules is presented. Nitrobenzene, was selected to represent the nitroaromatics, is detected via “turn-off” response and the limit of detection was found to be 350ppm. Pyridine, selected to represent nitroamines, was detected by a “turn-on” mechanism. The location and tautomeric/protolytic structures of fluorescein eluded characterization by powder X-ray diffraction and FT-IR. A quantitative method was developed and revealed that the relative amount of fluorescein incorporated was < 1% by weight, even at high doping level. Density functional theory (DFT) methods at the B3LYP/LAN2DZ level of theory supplemented experiment to better understand the molecular interactions within the system. Electronic spectroscopy and isothermal N2 sorption analysis supports the encapsulation of a distribution of doubly and singly charged anionic fluorescein within ZIF-8 pores. Salt metathesis and DFT reveal a strong coordination of fluorescein to Zn2+ ions via carboxylate function. The structural analog lacking this functionality, 6-hydroxy,9-phenyl, xanthen-3-one, was synthesized to gain insight on the role of the carboxylate. | Poster #: 66 Campus: California State University, Fresno Poster Category: Diagnostics/Imaging/Analytical Keywords: fluorescence, x-ray, blood Project Title: A phantom-based study of x-ray fluorescence detectability of iron and zinc in human cutaneous blood Author List: Mahajan, Vega ; Graduate, Physics, California State University, Fresno, Presenting Author
Gherase, Mihai; Faculty, Physics, California State University, Fresno Abstract Monitoring essential trace elements in the human body is an important part of clinical metabolic health assessment. Rapid, non-invasive, non-destructive, and low-dose monitoring of trace elements can be achieved by x-ray fluorescence (XRF) measurements. XRF detectability of iron (Fe) and zinc (Zn) in the superficial blood pool of the human skin was investigated in this study. The method is as an economical alternative to current clinical measurements using inductively coupled plasma mass spectrometry. Fe and Zn atomic absorption standard solutions, each with a concentration of 1001 mg/L were diluted to prepare six solutions each containing Fe in 0, 100, 200, 300, 400, and 500 mg/L concentrations and Zn in 0, 10, 20, 30, 40, and 50 mg/L concentrations, respectively. Solutions were poured into two different cylindrical cups of 0.5 mm and 0.9 mm wall thickness made of polyoxymethylene (POM) plastic (2.5 mm inner diameter and 5 cm length). In our previous work, POM was shown to mimic well the x-ray attenuation coefficient of human skin in the 5 to 15 keV x-ray photon energy range. Cylindrical wall thickness simulated in vivo variation of the combined blood, microvasculature, and skin x-ray attenuation. Spatially selective excitation of Fe and Zn was performed using a small x-ray beam (~1.5 mm) from an integrated x-ray tube and polycapillary x-ray lens system operated at 50 kV voltage, 1 mA current, and 1.8 mm aluminum filtration. Computer-controlled silicon x-ray detector with multichannel analyzer and two orthogonal positioning stages completed the experimental setup. An optimal grazing-incidence XRF method developed in our lab was used to acquire three 300-s x-ray spectra for each concentration. Spectral Kα peak area measurements at 6.4 and 8.6 keV energies yielded Fe and Zn calibration lines, respectively. Detection limit was computed as three times the peak area uncertainty at zero concentration divided by the calibration line slope. Fe and Zn detection limits of (23.5±0.6) mg/L and (3.16±0.02) mg/L for the 0.5 mm POM cylinder, and (80±7) mg/L and (6.6±0.7) mg/L for the 0.9 mm POM cylinder were obtained. Human blood concentration ranges reported in the literature are: 310 to 610 mg/L for Fe and 4 to 17 mg/L for Zn. Comparison between detection limits and human blood levels is encouraging. Radiation dose to skin and the effects of varying x-ray attenuation and blood volume on concentration measurements are targeted in future work. | Poster #: 67 Campus: San Diego State University Poster Category: Diagnostics/Imaging/Analytical Keywords: Zeptomole Sensitivity, COVID-19 N-Protein Detection, Multi-Photon Laser Wave Mixing Project Title: Novel Detection of SARS-CoV-2 Nucleocapsid and Spike Proteins Using Microfluidics and Nonlinear Multi-Photon Laser Wave-Mixing Spectroscopy Author List: Shatirishvili, Nino; Graduate, Chemistry and Biochemistry, San José State University, Presenting Author
Tong, William; Faculty, Chemistry and Biochemistry, San Diego State University, Presenting Author Abstract We report sensitive and selective detection of SARS-CoV-2 Nucleocapsid (N) and Spike (S) proteins, the biomarkers of COVID-19 using laser wave-mixing spectroscopy. Current N- or S-protein detection methods (non-antigen methods) require more time-consuming and complicated labeling steps and PCR for selective detection. Our patented nonlinear laser wave-mixing methods offer significant advantages including label-free native detection, excellent sensitivity (zeptomole), small sample requirements, short optical path length, high spatial resolution, and portable detector designs. The wave-mixing signal is generated when the two input beams are mixed inside the analyte and it can be collected with virtually 100% efficiency and excellent signal-to-noise ratios. The signal has a quadratic dependence on analyte concentration, and hence, small changes can be monitored more effectively. Since wave-mixing probe volume is small (nanoliter to picoliter), it is intrinsically suitable for microfluidics or capillary-based electrophoresis systems (e.g., 75 μm i.d. fused silica capillary). In addition, different biomarkers can be immobilized on a custom 3D-printed slide. Since wave mixing is an absorption-based method, both fluorophore and chromophore labels could be used, if desired. One can run a standard protein ladder to estimate capillary electrophoresis retention time for COVID-19 proteins. The glass slides and microarrays used in these detection methods suitable for proteins, biomarkers, etc., are custom 3D printed in our research lab. When using a compact UV laser, native label-free proteins could be detected at zeptomole levels without using PCR. Excellent detection sensitivity levels for PCR-free detection of CoV-2-N and CoV-2-S proteins are demonstrated using a Chromeo P540 label so that a compact 532 nm visible solid-state excitation laser can be used. | Poster #: 68 Campus: California State University, Los Angeles Poster Category: Disease (Pathogens) Keywords: Innate immunity, Tuberculosis, Mycobacteria Project Title: Development of a protocol to assess the antimycobacterial activity of lung epithelial cells Author List: Wesley-Cardwell, India; Undergraduate, Biological Sciences, California State University, Los Angeles, Presenting Author, Nagel Award Nominee
Abou Abbas, Dana; Graduate, Biological Sciences, California State University, Los Angeles
Dzul, Janette; Graduate, Biological Sciences, California State University, Los Angeles
Porter, Edith; Faculty, Biological Sciences, California State University, Los Angeles Abstract Mycobacterium tuberculosis (Mtb), an airborne pathogen, affects people on a global scale. Increasing bacterial resistance against antimycobacterial drugs and the limited efficacy of the current tuberculosis vaccine warrant novel approaches to combat tuberculosis. Epithelial cells lining the airways are the first to encounter Mtb. Epithelial cells increase the production of antimicrobials after stimulation with certain cytokines, proteins that are used in cell–cell communication. T helper cells of the subtype TH1 are specialized immune cells that are activated with the current tuberculosis vaccine leading to enhancement of macrophage mediated killing of Mtb. Our lab hypothesizes that incorporating responses of the subtype TH17 in tuberculosis vaccines may offer enhanced protection because TH17 cells are known to activate epithelial cells. As a first step, we aim to test the antimycobacterial activity of lung epithelial cells stimulated with TH17-derived cytokines using Mycolicibacterium smegmatis (Ms, formerly Mycobacterium smegmatis), an avirulent model organism for Mtb. Mycobacteria have lipid-rich cell walls that aid in aggregation to protect bacteria against lysis, making it unreliable to measure bacterial proliferation by colony-forming unit assays. Therefore, we decided to develop a nucleic acid-based assay. The objective of this study was to develop a protocol for lysing Ms, extracting its genomic DNA (gDNA) and quantifying the gDNA with SYBR Green chemistry polymerase chain reaction (qPCR). Ms incubation with Tri-reagent combined with intermittent high-speed mixing with 0.1- and 0.5-mm glass beads liberated bacterial gDNA which was purified with Direct-zol™ DNA/RNA Miniprep. Primers specific for Ms were designed and a qPCR protocol showing gDNA input dependent and Ms specific SYBR green fluorescence amplification was developed. This protocol was then used to quantify Ms proliferation in secretions collected from the lung derived epithelial cells A549 after treatment with TH17 cytokines or solvent control. Preliminary data suggest that some cytokines may induce antimycobacterial activity in A549 cells. This research may inform future tuberculosis vaccine design that incorporates TH17 and lung epithelial cell responses and may aid in the battle against tuberculosis. This work was supported by NIH R25GM061331, NIH T34GM08228, CSUPERB Microgrant, and CalStateLA Provost’s Research Restart Grant. | Poster #: 69 Campus: California State University, Los Angeles Poster Category: Disease (Pathogens) Keywords: TH17 cells, Tuberculosis, Epithelial cells Project Title: Antimicrobial Effector Molecule Response in Alveolar Type II Cells Using Pro-Inflammatory Cytokines Author List: Abou Abbas, Dana; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Soriano, Rachelle; Undergraduate, 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 main cause of tuberculosis (TB), which primarily affects the lungs. Globally, TB is considered the second greatest infectious disease-related cause of mortality to this date. This is in part due to the rising prevalence of multidrug-resistant Mtb strains and poor efficacy of the existing TB vaccine. As a result, novel methods for combating Mtb must be developed. The current vaccine triggers responses of TH1 cells, a subset of lymphocytes that activate macrophages via the cytokine IFNg. However, epithelial cells, among them alveolar cells, are one of the first in line to come in contact with inhaled Mtb. In response to being stimulated with microbial products and certain cytokines such as IL1b, epithelial cells increase their production of antimicrobial peptides and lipids. Our lab pursues the concept that lung epithelial cells stimulated with cytokines produced by the T helper cell subset TH17, namely IL17 and IL22, increase their antimycobacterial activity and that incorporating TH17 cells responses in the design of novel vaccine against Mtb could afford better protection against TB. We hypothesize that stimulating A549 cells, alveolar Type II-like cells, with cytokines produced by TH17 cells will increase their production of antimicrobial effector molecules and antimycobacterial activity. The objective of this study was to test the antimicrobial response of alveolar type 2 like cells to TH17 cytokines in vitro. We stimulated A549 cells with the TH17 cytokines IL17 and IL22 in triplicate wells and measured intracellular accumulation of the antimicrobial peptide HBD2 using immunofluorescence and nuclear counterstain with DAPI. DAPI was analyzed via ImageJ and HBD2 was enumerated by three independent observers. Overall lipid secretion was assessed with the lipophilic dye Nile red. Stimulation with the pro-inflammatory cytokines IL1b and IFNg was included as reference. Using One Way ANOVA, we observed significant differences for the number of DAPI events and HBD2 + cells/100 DAPI events (p < 0.001). Using posthoc analysis with Bonferroni a statistically significant difference between control and IL1b and IL17/IL22 treatment (p < 0.001 and p = 0.018, respectively) was seen, though the Nile Red data were inconclusive. This research may govern future vaccine design for TB. This work was supported by a CSUPERB Microgrant and Cal State LA Provost’s Research Restart Grant.
| Poster #: 70 Campus: California State University, San Bernardino Poster Category: Disease (Pathogens) Keywords: Macrophage, Feline Coronavirus, Cell Line Project Title: Generation of Feline and Cheetah Cell Lines to Study Feline Coronavirus Author List: Espinoza, Adam ; Graduate, Biology, California State University, San Bernardino, Presenting Author
Fraser, Cameron; Graduate, Biology, California State University, San Bernardino, Presenting Author
Newcomb, Laura ; Faculty, Biology, California State University, San Bernardino
Abstract Feline Coronavirus (FCoV) typically causes a subclinical enteric infection in domestic cats, with ~5-10% of infections resulting in virulent feline infectious peritonitis, where viruses replicate within macrophages and disperse throughout the host body. However, in the 1980s, an outbreak of FCoV infected captive cheetahs and resulted in 60% mortality within 3 years. Cheetahs suffered multiple bottlenecks and have little genetic diversity, leading to increased susceptibility to viral pathogens such as FCoV. However, tools to study FCoV infection in vitro are lacking, with only one macrophage domestic feline cell line, notorious for difficult propagation, available from the American Tissue Culture Collection (ATCC), and no large feline cell lines. To create a domestic feline immortalized macrophage cell line, domestic feline whole blood was purchased and monocytes isolated using Lymphoprep gradient. Adherent cells were transduced with lentivirus encoding SV40-Large T-antigen for immortalization. Cells were diluted to single cell and expanded to obtain clonal cell populations. Colonies were passaged and doubling rates taken, demonstrating doubling times within 24 hours. Total RNA was isolated and reverse transcription qPCR confirmed mRNA expression of SV40-Large T-antigen, along with CD13, the macrophage receptor for FCoV, and CD4, the macrophage receptor for Feline Immunodeficiency Virus (FIV). Selected immortalized clonal cell populations, named FMac, were cryopreserved and are being further tested to confirm macrophage properties. We examined FCoV infection in FMac compared to Crandell-Rees Feline Kidney Cell (CRFK). Our results revealed no cytopathic effect (CPE) in FMac. Yet, tissue culture infectious dose assays (TCID50) show viral replication occurred in FMac cell line. Continued characterization of FMac will include additional gene expression analysis, assessment of macrophage phagocytosis, and reproducibility of initial FCoV infection evaluation. After characterization, cell lines will be deposited with ATCC. Access to a rapidly growing domestic feline macrophage cell line will allow researchers to address questions regarding host-virus interactions. We will use this protocol to isolate and immortalize macrophages from cheetah whole blood samples to be obtained from our collaborators at The Living Desert Zoo (Palm Desert, CA). Funding for this project was provided by Edison STEM-NET to CF and CSUSB-Office of Student Research to AE. | Poster #: 71 Campus: California State University, Fullerton Poster Category: Disease (Pathogens) Keywords: Mitochondria , Parasites, Ion channels Project Title: Establishing the role of the mitochondrial Voltage-Dependent Anion Channel (VDAC), in Trypanosoma cruzi Author List: Poon, Kyle; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Mesones, Sebastian; Graduate, Biological Science, California State University, Fullerton
Jimenez, Veronica; Faculty, Biological Science, California State University, Fullerton
Abstract The protozoan Trypanosoma cruzi is the causative agent of Chagas Disease, a neglected tropical disease that affects millions globally and has no effective treatment or preventive vaccine. As it transitions from insect vector to human host, T. cruzi faces many homeostatic challenges, including oxidative stress, fluctuations in osmolarity, and nutrient availability. Ion channels regulate the parasite’s ability to respond to environmental stressors. Our lab has previously identified a mechanosensitive ion channel located in the contractile vacuole that, when knocked out, leads to reduced infectivity, irregular morphology, and impaired motility in the parasite. RNA-seq data from the cell line with the ablated channel showed that Voltage-Dependent Anion Channel (VDAC) transcripts were significantly downregulated, suggesting a functional link between the two proteins. VDAC is a highly expressed outer mitochondrial membrane protein that, in other eukaryotes, has been shown to play a part in regulating cell death, mitochondrial morphology and metabolism. However, the function of VDAC in T. cruzi remains uncharacterized. To elucidate the function of this channel in the parasites, a conserved sequence was targeted using CRISPR/Cas9 strategies to generate a VDAC-deficient cell line. The mutant strain exhibited significant growth defects relative to the wild-type, demonstrating that VDAC is critical for parasite replication. Fluorometric analysis indicates that reduced VDAC expression results in depolarization of the mitochondria confirming the role of VDAC in maintaining mitochondrial membrane potential. We are further interrogating the physiological function of the channel by measuring mitochondrial calcium uptake, ATP production, and reactive oxygen species generation. Improving the understanding of VDAC’s role in T. cruzi would help guide future drug development. | Poster #: 72 Campus: San José State University Poster Category: Disease (Pathogens) Keywords: intercellular junctions, Streptococcus pneumoniae, Python Project Title: Measuring Intercellular Junction Organization During Microbial Infection With a New Python Algorithm Author List: Mo, Devons; Graduate, Biological Sciences, San José State University
Homez, Nicole; Undergraduate, Biological Sciences, San José State University
Nguyen, Vivian; Undergraduate, Biological Sciences, San José State University, Presenting Author
Do, Jasmin; Undergraduate, Biological Sciences, San José State University, Presenting Author
Jakoush, Tarek; Graduate, Biological Sciences, San José State University
Zaroo, Suhanee; Undergraduate, Biological Sciences, San José State University
Rosa-Cortes, Juan; University of Puerto Rico
Hasan, Shakir; University of Toronto
Xu, Shuying; Tufts University
Adams, Walter; Faculty, Biological Sciences, San José State University Abstract Many infectious microbes compromise our respiratory tract during lung infection by disrupting intercellular junctions, including adherens junctions and tight junctions. Both adherens junctions and tight junctions are important protein structures that are responsible for maintaining intercellular integrity, so their disruption could lead to the development of severe disease by a pathogen. One such pathogen is Streptococcus pneumoniae, a leading cause of pneumonia, which can successfully breach the epithelial barrier and cause severe infections such as septicemia and meningitis. Fluorescence microscopy analysis of intercellular junction protein disruption by invasive bacteria and viruses has yielded major advances in our understanding of their pathogenesis. Unfortunately, a lack of automated image analysis tools that can tolerate substantial variability that exists in sample-to-sample staining has limited the accuracy in evaluating intercellular junction organization quantitatively. We have created an open source, automated Python computer script called “Intercellular Junction Organization Quantification” or IJOQ that can handle a high degree of sample-to-sample staining variability and robustly measure intercellular junction integrity. In silico validation of IJOQ was successful in analyzing computer generated images containing varying degrees of simulated intercellular junction disruption. Accurate IJOQ analysis was further confirmed using images generated from in vitro and in vivo bacterial infection models. When compared in parallel to a previously published, semi-automated script used to measure intercellular junction organization, IJOQ demonstrated superior analysis for all in vitro and in vivo experiments described herein. These data indicate that IJOQ is an unbiased, easy-to-use tool for fluorescence microscopy analysis and will serve as a valuable, automated resource to rapidly quantify intercellular junction disruption under diverse experimental conditions. | Poster #: 73 Campus: California State Polytechnic University, Pomona Poster Category: Disease (Pathogens) Keywords: mice, Candida albicans, Cytokines Project Title: Comparing the effects of Sex and CB2R between uninfected and Mice with a Systemic Candida albicans infection Author List: Henriquez, Diego; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Nadales, Nathalie; Graduate, Biological Sciences, California State Polytechnic University, Pomona
Buckley, Nancy; Faculty, Biological Sciences, California State Polytechnic University, Pomona
Abstract Candida albicans, an opportunistic pathogen found in our normal flora, is the fourth most common nosocomial infection in the United States. ICUs across the US report C. albicans responsible for 10% of blood infections encountered. Incidentally, individuals seeking symptomatic relief resort to the use of marijuana and its compounds such as THC and CBD. These marijuana compounds interact with the individuals’ endocannabinoid system (ECS). The ECS includes cannabinoid receptors (CBRs). The best known CBRs are the central and peripheral cannabinoid receptors, CB1R and CB2R, respectively. CB1R is predominantly expressed in the CNS while CB2R is mostly expressed in cells of the immune system. Thus, it is important to understand the role of CB2R and how it may affect fungal resistance. Another factor affecting susceptibility to fungal infections is sex. We have found that male mice tend to be more susceptible to C. albicans infections.
To investigate the role of sex and CB2R in the resistance to C. albicans infection, CBR2 knockout and wild type male and female mice were intravenously injected with nothing or with 5 uL/g of 7.5x10^6 C. albicans cells/mL. Kidneys, blood, spleen, and bone marrow were collected 3 days after the infection. Kidneys were analyzed for fungal load and cytokine (IL-6 and TNF-α) levels were assessed from kidneys and blood serum. The spleens and bone marrow were pooled by sex and genotype (F +/+, F-/-, M+/+, M-/-). Splenocyte cytokine production was determined by Enzyme Linked Immunosorbent Assays (ELISA). Neutrophil content was determined from bone marrow cells using flow cytometry.
There was no difference in kidney fungal load between the sexes nor genotypes investigated. Serum IL-6 was absent in uninfected mice, but present in all infected mice. IL-6 levels were similar between sexes and genotypes. Male kidneys have higher IL-6 and TNF-α levels compared to the other groups. The percentage of bone marrow neutrophils was higher in infected mice and males had a slightly higher level of neutrophils post infection. These results provide baseline data from which we will pursue experiments in which mice will be treated with CBD to investigate its effects on mouse resistance to systemic C. albicans infection.
| Poster #: 74 Campus: San Francisco State University Poster Category: Disease (Pathogens) Keywords: 16S ribosomal gene PCR sequencing , Brown Blotch disease , Pseudomonas Project Title: Identification of Pathogenic Bacteria on White Button Mushrooms with Brown Blotch Disease Author List: Badala, Yogesh ; Graduate, Biology, San Francisco State University, Presenting Author
Quach, Sammy; Graduate, Biology, San Francisco State University, Presenting Author
Chen, Lily; Faculty, Biology, San Francisco State University
Abstract The white button mushroom Agaricus bisporus (A.bisporus) is one of the most widely cultivated mushrooms in the United States because of its taste and nutritional value. However, it is also more prone to microbial growth. Bacterial pathogens, including Pseudomonas tolaasii that releases a pore-forming toxin called tolaasin, are responsible for several detrimental mushroom diseases, including cobwebs, soft rot, cavities, and brown blotches*. This study analyzed the microbial diversity of the button mushroom by examining variables of sampling locations, nutrient requirements, incubation temperature, and time. The bacterial isolates obtained from the fruiting bodies of fresh A.bisporus were identified by morphological and molecular characteristics using different biochemical tests and culture media. The 16S ribosomal gene PCR sequencing reactions were performed to identify the isolates down to the genus and species level. Thirteen isolates were characterized, and seven isolates were found belong to the Pseudomonas genus that may be involved in the Brown Blotch disease (including Pseudomonas tolaasii). In addition, Ewingella americana, a new member of the Enterobacter that rarely causes human infection but has the ability to induce symptoms of internal stipe necrosis in mushroom was obtained. Ongoing studies will assess pathogenicity and host ranges of the bacterial isolates on edible mushroom (Kosh’s Postulates) and characterize pathogen-helper bacteria interactions.
*Reference: Hermenau, R., Kugel, S., Komor, A. J., & Hertweck, C. (2020). Helper bacteria halt and disarm mushroom pathogens by linearizing structurally diverse cyclolipopeptides. Proceedings of the National Academy of Sciences, 117(38), 23802-23806.
| Poster #: 75 Campus: California State University, Long Beach Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: atherosclerosis, innate immunology, macrophage Project Title: Investigating the role of Autophagy in C1q-Mediated Modulation of the Macrophage Inflammasome Author List: Smith, Lydia; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author, Nagel Award Nominee
Sinatra, Jade; Undergraduate, Biological Sciences, California State University, Long Beach
Fraser, Deborah A.; Faculty, Biological Sciences, California State University, Long Beach
Abstract Atherosclerosis is a chronic inflammatory disease which is characterized by lesional macrophage digestion of oxidized low density lipoproteins (oxLDL). Macrophages release pro-inflammatory cytokines such as IL-1β through activation of the NLRP3 inflammasome, which often leads to exacerbation of inflammation and disease progression. We have previously determined that the innate immune protein C1q plays a protective role in the early stages of atherosclerosis by modulating and dampening components of the NLRP3 inflammasome. C1q effectively polarizes macrophages toward an anti-inflammatory phenotype, thereby decreasing the secretion of pro-inflammatory cytokine IL-1β. During modified lipoprotein clearance, C1q also facilitates the activation of autophagy markers on macrophages through the natural degradation and recycling of cellular elements. Recently, the activation of autophagy in macrophages has been shown to play a central role in regulation of the NLRP3 inflammasome by acting in an atheroprotective manner to increase macrophage survival. Using this information, we tested the hypothesis that C1q dampens the inflammasome through activation of autophagy. Human monocyte derived macrophages (HMDM) were cultured from whole blood, and treated in either the presence or absence of 3-MA autophagy inhibitor. The cells were then treated with oxLDL +/- C1q, which was followed by collection of mRNA and cell supernatant. Gene expression of NLRP3 and IL-1β were measured by quantitative PCR, and protein levels of IL-1β were measured using Luminex multiplex assay. Our data confirmed that, as was previously observed, C1q dampened IL-1β gene and protein expression in M1 inflammatory macrophages. However, this C1q-mediated reduction in IL-1β was not seen in the presence of autophagy inhibitor 3-MA. These data suggest that C1q is dampening the inflammasome, at least partially, through the activation of autophagy. This provides a novel mechanism through which C1q may be beneficial in atherosclerosis.
This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number SC3GM111146 (DF). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
| Poster #: 76 Campus: California State University, Northridge Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Three-Dimensional Models, Pancreatic Cancer, Tumor-Stroma Interactions Project Title: Molecular Analyses of Multicellular Pancreatic Cancer Spheroids Author List: Todd, Audrey; Undergraduate, Biology, California State University, Northridge, Presenting Author, Nagel Award Nominee
Aquino, Albert; Graduate, Biology, California State University, Northridge
Kelber, Jonathan; Faculty, Biology, California State University, Northridge
Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer. Mesenchymal stem cells (MSC’s) present in the PDAC microenvironment have been reported to contribute to therapy resistance – a major clinical problem in this malignancy. Prior work has demonstrated that PDAC/MSC multicellular spheroids are significantly more compact that PDAC mono-culture spheroids. Furthermore, the tumor cells within these multicellular spheroids show a significant upregulation of cell dormancy markers suggesting that this may be a very important model for understanding mechanisms by which PDAC tumor cells acquire therapy resistance within the primary tumor. Here, we employ a 3D spheroid co-culture model of PDAC cells and MSCs to analyze the expression changes for a panel of genes that are relevant within the primary tumor cells and stromal MSCs. By combining human PDAC cells and mouse MSCs into our system, we can use species-specific qPCR primers to quantify gene expression profiles in each cell type. When in 3D co-culture, PANC1 cells showed an increase in expression of epithelial and mesenchymal markers, cell stress markers, and a mix of tumor dormancy and dormancy recovery markers. In contrast, the C3H10T1/2 MSCs showed significantly higher expression of cancer cell stemness markers. Additionally, 3D spheroids of MiaPaCa2/MSCs caused MiaPaca2 cells to express decreased levels of dormancy genes and increased expression of epithelial genes. These co-cultured MSCs expressed significantly increased expression of cell stress markers and decreased cellular proliferation markers. Notably, Inhba (the gene coding for the activinA protein) was significantly increased in the MSCs under co-culture with both the PANC1 and MiaPaca2 cells. This is noteworthy since our group has previously identified activinA as a factor within the tumor microenvironment to promote therapy resistance. Therefore, we tested whether Follistatin, a naturally-ocurring inhibitor of activinA, could block any MSC-induced effects in these 3D multicellular spheroids. While Follistatin did not seem to affect the ability of MSCs to induce spheroid compaction, we are still testing its effects on gemcitabine sensitivity and spheroid expansion. Taken together, these studies and our findings to date hold the potential to develop new strategies for sensitizing PDAC cells. | Poster #: 77 Campus: California State University, Chico Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: skin, stem cells, wounds Project Title: Mesenchymal stem cell (MSC) conditioned media facilitates wound healing Author List: Rueb, Kristen; SpecBio
Payne, Madison; Undergraduate, Biological Sciences, California State University, Chico
Stachura, David; Faculty, Biological Sciences, California State University, Chico, Presenting Author
Abstract Mesenchymal stem cells (MSCs) are adult stem cells that can self-renew and differentiate into myocytes, chondrocytes, adipocytes, and osteoblasts. These cells have demonstrated therapeutic potency for treating numerous clinical conditions, including a multitude of inflammatory diseases, ischemia, fibrosis, myocardial infarction, type I diabetes, and autoimmune diseases. Importantly, MSCs can also be used for a multitude of regenerative medicine procedures. MSCs reside in numerous tissues of the body, and the most easily obtained are adipose-derived MSCs (ADMSCs) from lipoaspirates obtained during cosmetic surgery. Evidence indicates that the main beneficial effects of MSCs are mediated by the growth factors and cytokines that they secrete. Thus, collecting the media after MSCs are cultured, known as conditioned media (CM), captures the multitude of proteins that potentially have therapeutic value. To assess if CM possesed wound-healing properties, we cultured MSCs and collected the proteins that they secreted, optimizing their growth to produce CM with high levels of reparative growth factors and cytokines. CM not only increased the rate of proliferation of human keratinocytes, but in vitro scratch assays on skin cells also indicated that CM accelerated wound repair in a dose-dependent manner. Additionally, we performed in vivo regeneration assays by cutting the caudal fins of zebrafish, which regenerate autonomously, and adding increasing amounts of CM to the cultures. Overall, we witnessed an increase in wound repair and fin regeneration. These experiments indicate that MSC CM effectively accelerates wound healing and is a potential treatment modality for a variety of human wounds.
| Poster #: 78 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: alcohol, Drosophila, epigenetic Project Title: Transcript-specific effects of developmental ethanol exposure on the expression of chromatin-modifying genes in Drosophila Author List: Marsh, Joshua; Graduate, Biological Sciences, San José State University, Presenting Author
Filardo, Desirée; Graduate, Biological Sciences, San José State University, Presenting Author
French, Rachael; Faculty, Biological Sciences, San José State University
Abstract Ethanol is a teratogen; developmental alcohol exposure (DAE) in humans causes a variety of deleterious phenotypes, including slow growth, metabolic changes, behavioral difficulties, and intellectual disabilities. Recent findings in mammals indicate that metabolic changes associated with DAE involve long-term changes in gene expression mediated by epigenetic effects.
We have established Drosophila as a model for DAE. Using this model, we have found that DAE in flies causes phenotypes similar to those seen in mammals exposed during fetal development, including developmental delay, reduced adult size, smaller brains, CNS dysfunction, impaired insulin signaling and lipid metabolism, and reduced survival. Some of these phenotypes, including impaired lipid metabolism and sedation resistance, persist into adulthood. Consistent with this observation, DAE in flies causes long term changes in gene expression.
Ethanol exposure in adult flies alters the expression of histone modifying enzymes. We hypothesize that some of the long-term changes in phenotype and gene expression caused by DAE are due to epigenetic alterations in gene expression due to similar effects on the expression of histone modifiers. We therefore tested the DAE sensitivity of flies mutant for a variety of histone modifiers. In addition, we used qPCR to measure the expression of genes encoding histone modifiers in ethanol-reared larvae.
We will present data showing that mutations disrupting the histone modifiers Sirt1, Lid, dG9a, JHDM2, NO66, and Nejire (Nej) result in changes in sensitivity to DAE. Further, DAE causes reduced expression of G9a, Sirt1, and lid in larvae, and specifically upregulates at least one nej transcript. Thus, DAE alters epigenetic regulation of gene expression, and it is likely that some DAE-induced phenotypes are due to these effects.
We will present the results of experiments to test the effects of DAE on additional histone modifying enzymes, as well as the role of those proteins in sensitivity to developmental ethanol. We are also testing whether persistent DAE-induced phenotypes are due to changes in epigenetic regulation of gene expression. This research was supported by grants from the National Institutes of Health National Institute of General Medical Sciences (5SC3GM103739) and National Institute on Alcohol Abuse and Alcoholism (1R15AA027678), and a Project Development Grant from the California State University Program for Education and Research in Biotechnology.
| Poster #: 79 Campus: California State University, Fullerton Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: glioblastoma multiforme, Lipidomics, bioinformatics Project Title: Lipidomic Analysis of Ceramide Synthases in Human U251 Cells Resistant and Non-Resistant to Temozolomide Treatment For Glioblastoma Multiforme Author List: Weng, Xiaohui; Graduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
da Silva Rosa, Simone; Postdoc, Department of Human Anatomy and Cell Science, Max Rady College of Medicine, University of Manitoba
Ghavami, Saeid ; Department of Human Anatomy and Cell Science, Max Rady College of Medicine, University of Manitoba
Pecic, Stevan; Faculty, Chemistry and Biochemistry, California State University, Fullerton Abstract Glioblastoma multiforme (GBM) is a type of primary brain cancer in the central nervous system (CNS) with a low median survival of 15 months. Currently, the only FDA-approved chemotherapeutic agent for the treatment of GBM is temozolomide (TMZ), an alkylating agent that initiates apoptosis. There has been evidence suggesting a link between ceramide synthases and the bcl2 family proteins, which could either promote or inhibit apoptosis. The long-term goal of our lab is to understand how the up- and downregulation of lipids, more specifically, the ceramide synthases, are correlated with the development of chemoresistance as a result of TMZ-induced autophagy in GBM patients.
Lipidomic analysis was used to compare the lipid distribution between TMZ-resistant and non-resistant U251 cells that were treated with control. A total of 30 lipids from 5 ceramide lipid subfamilies, each with 6 different chain lengths, were analyzed using ANOVA, pair-wise comparison, heatmap, volcano plot, and VIP score. These analyses were used to identify and visualize the lipids that are up- or down-regulated in the resistant and non-resistant U251 cells.
The heatmap showed that 8 out of 30 lipids were downregulated in the resistant cells, while the other 22 lipids were upregulated. Pair-wise comparisons from ANOVA identified at least one lipid from each ceramide subfamily that shows a statistically significant change in the resistant cells compared to the non-resistant cells. The volcano plot identified 4 lipids that are upregulated in the resistant cells.
The results indicate that some of the lipids from the ceramide family are up- or down-regulated in the GBM cells suggesting their involvement in the mechanism of TMZ resistance. Therefore, intervention in the synthesis pathways of these specific lipids could provide therapeutic benefits.
This study's results are significant because they help provide insights into the lipid profiling of U251 cells resistant to TMZ treatment for GBM. Furthermore, it also presents specific lipids as potential drug targets for combination therapy to enhance the efficacy of TMZ treatment and therefore prolong the survival time of patients. Our next step is to perform lipidomic analysis and compare the lipid profiling of TMZ-resistant and non-resistant U251 cells that are treated with control and TMZ treatment. | Poster #: 80 Campus: California State University, Los Angeles Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: TGF-beta , Smad2, Drosophila Project Title: Investigating if linker phosphorylation plays a role in dSmad2 signal duration and degradation Author List: Flota, Pablo; Graduate, Biological Sciences , California State University, Los Angeles, Presenting Author
Guanzon, John; Undergraduate, Biological Sciences , California State University, Los Angeles, Presenting Author
Castro, Kenny; Graduate, Biological Sciences , California State University, Los Angeles
Eivers, Edward ; Faculty, Biological Sciences , California State University, Los Angeles Abstract The Transforming growth factor β (TGF-β) signals are known to be important for cell proliferation, differentiation and cell fate determination during embryonic development and tissue homeostasis in adults. The TGF-β signaling family is divided into two primary pathways; the Activin/TGF-β pathway and Bone Morphogenetic Protein pathway (BMP). In this study we focused on the Drosophila TGF-β/Activin pathway, in particular how the transcription factor, dSmad2, is degraded after C-terminal phosphorylation (C-terminal phosphorylation leads to pathway activation). Most of what we know about the termination and degradation of Smad2 proteins has come from studies in mammalian cells/tissues. Others have demonstrated that mammalian Smad2 after activation is linker phosphorylated, ubiquitinated by the E3-ligase Nedd4L and, then degraded by cellular proteasomes. Our understanding of what happens to dSmad2 after C-terminal activation is limited, we and others have found that C-terminal phosphorylation leads to bulk degradation of dSmad2 proteins, but the mechanism is unknown. Here we will present data showing that mutation of dSmad2 linker sites into non-phosphorylatable alanines followed by mis-expression in Drosophila wing imaginal discs leads to adult wings with profound phenotypic and size changes, when compared to controls. We will also show that linker phosphorylation and cellular proteasomes do not seem to play a role in dSmad2 degradation. Interestingly, we find that when dSmad2 is C-terminally phosphorylated, at least four distinct cleavage products are visible below the full length dSmad2 protein on western blots. These findings suggest that a alternative mechanism to degrade dSmad2 proteins occurs in Drosophila. Currently we are investigating a number of potential proteases that may give us some insight into what cleaves dSmad2 after C-terminal phosphorylation. | Poster #: 81 Campus: California State University, Chico Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Diabetes, micropeptides, fatty acids Project Title: Differential Expression of Mitoregulin Could Decrease Long-Chain Fatty Acid Beta-Oxidation in Pancreatic Beta Cells Author List: Upton, Cody; Graduate, Biology, California State University, Chico, Presenting Author
Keller, David; Faculty, Biology, California State University, Chico Abstract There is currently no cure for Type 2 Diabetes and the exact cellular mechanisms involved in its development are poorly understood. Over 50 diabetes-associated genes have been identified, many of which reside in non-coding regions of the genome. Recent advances in genome sequencing have revealed that large sections of the genome labeled as long non-coding RNA (lncRNA) actually code for very small proteins called micropeptides. Micropeptide dysregulation has been associated with multiple diseases, but this has been understudied in Type 2 Diabetes. A lncRNA called LINC00116 has been shown to code for a micropeptide called Mitoregulin (Mtln). Mtln enhances beta-oxidation of long-chain fatty acids in muscle, heart, and fat cell mitochondria. Mtln has never been studied in beta-cells, which share many physical and chemical properties with muscle and heart cells. We hypothesized that Mtln is naturally expressed in beta-cells and increases the amount of fatty acid beta-oxidation. Rat insulinoma (INS-1) cells were used as a model for human pancreatic islets. We designed primers using the rat genome Mtln coding sequence (CDS) and performed reverse transcriptase quantitative PCR (qRT-PCR) using RNA from INS-1 cells to test for the expression of endogenous LINC00116. The results showed, for the first time, that INS-1 cells endogenously express LINC00116. We transformed Invitrogen Top10 E. coli with pcDNA 3 2x FLAG plasmid containing the Mtln CDS and isolated the cloned plasmid DNA. To study the exogenous expression of Mtln, we transfected HEK293 cells with the cloned plasmid DNA and performed Western blotting. We demonstrated that Mtln can be exogenously expressed in INS-1 cells. We performed MTS assays to investigate how Mtln overexpression affects fatty acid beta-oxidation in INS-1 cells. However, this did not show significant changes to INS-1 cell metabolism in high fatty acid conditions. We also performed Western blotting with an Anti-Mtln primary antibody to investigate the endogenous expression of Mtln in INS-1. This revealed another novel finding: Mtln is naturally expressed in INS-1 cells. We concluded that Mtln is endogenously expressed in human pancreatic beta-cells and that a knockdown in Mtln expression could hinder fatty-acid beta-oxidation and be a contributing factor to Type 2 Diabetes formation.
| Poster #: 82 Campus: California State University, Northridge Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: antibiotic resistance, RpoS, gene regulation Project Title: Developing an assay to assess biomolecules designed to interfere with activation of RpoS, a crucial regulator of stress response pathways Author List: Gutierrez , Andrea ; Undergraduate, Biology, California State University, Northridge, Presenting Author
Meza, Mayra; Undergraduate, Biology, California State University, Northridge, Presenting Author
Takahashi, Melissa; Faculty, Biology, California State University, Northridge
Abstract Antibiotic resistance is a serious worldwide issue that has been leading to more persistent infections, increasingly higher medical costs, and a greater number of fatalities. RpoS is a central transcriptional regulator that controls the expression of different stress response pathways in Escherichia coli. DsrA is a small RNA that binds to a region of the RpoS mRNA, leading to a change in conformation that facilitates the attachment of the ribosome, thereby activating the translation of RpoS. Therefore, by designing a molecule that binds to DsrA we can disrupt the bacteria from defending themselves against multiple stress factors such as antibiotics. To accomplish this, we developed a gene expression assay that allowed us to distinguish between the cells that were expressing RpoS (ON) and those that were not expressing the protein (OFF). We fused the 5’ untranslated region of the rpoS gene to the green fluorescence protein (GFP) coding sequence and cloned it on a plasmid. We also cloned the sRNA DsrA and an antisense RNA, antiDsrA, which binds to DsrA and prevents the translation of RpoS, into separate plasmids. The E. coli cells that contain the DsrA and RpoS plasmids glow green. However, in the presence of a molecule that binds to DsrA the cells do not fluoresce because the RpoS-GFP translation cannot be initiated. To be able to easily distinguish between the OFF and ON conditions, we optimized the promoter strength for each plasmid. We tested different RpoS and DsrA promoters to obtain the most effective activation. Then, we tested for different antiDsrA promoters that could be strong enough to block the translation of RpoS by binding to DsrA. With the completion of this expression assay, we demonstrated that DsrA can bind to RpoS and induce its translation and antiDsrA can adhere to DsrA and suppress the expression of RpoS. Our next step is to employ this assay to test peptides that will obstruct the expression of RpoS, targeting the antibiotic resistance of E. coli. This research was supported by the Takahashi Lab start-up funds and NIH grant #SC2GM136500 and BUILD PODER NIH grant #RL5GM118975 and #UL1GM118976. CSUN’s College of Science and Mathematics Office of the Dean additionally supports Andrea Gutierrez. | Poster #: 83 Campus: California State University, Los Angeles Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: hyphal transition, Candida albicans, Yeast Casein Kinase Project Title: Role of Yeast Casein Kinase 2 in Hyphal Regulation via the Transcription Repressor NRG1 in Candida albicans Author List: Tzarax, Alex; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Abstract Candida albicans is an opportunistic pathogen in patients with immunocompromised conditions (e.g., AIDS). One of the virulence factors of C. albicans is its ability to transition morphology between yeast, pseudohyphae, and true hyphae. Inability to transition its morphology dramatically reduces its capability to invade epithelial and endothelial tissues in murine models. A previous study shows that the C. albicans YCK2 deletion mutant strain (yck2Δ strain) was locked in pseudohyphae, had a lower growth rate, and dysregulated UME6 transcription (a transcriptional regulator of yeast to hyphal program). Additionally, transcriptional analysis shows that TUP1 and NRG1 form a complex to repress the transcription of genes like UME6, which turns on the yeast to hyphal program, were downregulated in the yck2Δ strain. This finding suggests that C. albicans Yeast Casein Kinase 2 (CaYck2p) function may affect the transcription of NRG1 and TUP1, which repress the transcription of hyphal specific genes (HSGs). We hypothesized that CaYck2p is involved in the transcription of hyphal repressor gene NRG1 and that the overexpression of NRG1 will restore morphology and growth defect of the yck2Δ strain. We aimed to overexpress the hyphal repressor gene NRG1 in the yck2Δ strain and asses if defective phenotypes are restored compared to the wild type. A constructed recombinant DNA containing a strong promoter of the ADH1 gene attached to the open reading frame of the NRG1 gene was integrated into C. albicans genome. Then, RNA extraction was performed, followed by cDNA and real-time PCR with SYBR green technology probing for target genes NRG1 and UME6 and the housekeeping gene ACT1. This study showed that overexpression of NRG1 can repress the dysregulated transcription of UME6 and restore cell morphology and growth defect of the yck2Δ strain, which suggests that Yck2p inhibits hyphal formation by activating the NRG1 gene. Since overexpressing NRG1 in the yck2Δ strain was able to restore the defective morphology, growth, and repress dysregulated UME6 transcription, Yck2p seems to inhibit hyphal formation by activating the hyphal repressor NRG1. Understanding the pathways involved in morphology transition could lead to the creation of novel fungus treatments, including C. albicans. | Poster #: 84 Campus: California State University, Fullerton Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: sRNA, Sinorhizobium meliloti, symbiosis Project Title: Assessing whether SmelC506 and SmelC625 genes are transcriptionally regulated by the ExoS/ChvI signaling pathway in Sinorhizobium meliloti Author List: Valdivia, Jacqueline; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Valencia, Caleb; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Chen, Esther; Faculty, Biological Science, California State University, Fullerton
Abstract Bacteria-plant symbiotic relationships are relevant to sustainable agriculture and combating climate change caused by industrial fertilizer manufacturing. In particular, Sinorhizobium meliloti is a bacterium that fixes atmospheric nitrogen into an accessible form for its plant host, Medicago sativa; in return, S. meliloti receives carbon sources. This relationship is possible due to the bacteria’s ExoS/ChvI two-component signaling pathway that transcriptionally regulates genes essential for symbiosis. Previous studies identified 28 small RNA (sRNA) genes that ExoS/ChvI potentially regulates. sRNAs are small non-protein encoding RNAs that post-transcriptionally regulate gene expression by interacting with mRNA transcripts. Our research aims to determine if the ExoS/ChvI pathway transcriptionally regulates two sRNA genes, SmelC506 and SmelC625. To determine whether ExoS/ChvI regulates these sRNA genes, transcriptional fusion alleles were constructed by fusing the DNA region upstream of the sRNA gene to the GUS reporter gene. These transcriptional fusion alleles were transduced into three S. meliloti strains: wild-type, a chvI partial-loss-of-function mutant, and a chvI gain-of-function mutant. If an sRNA gene were regulated by ExoS/ChvI, we expected to observe a reciprocal change in GUS activity in both chvI mutant strains compared to in the wild-type. Preliminary results for the SmelC506 transcriptional fusion showed higher GUS activity in the chvI gain-of-function mutant and slightly lower GUS activity in the chvI partial-loss-of-function mutant compared to the wild-type. These results indicate that SmelC506 is positively transcriptionally regulated by chvI. In contrast, the SmelC625 transcriptional fusion showed higher GUS activity in both chvI mutants compared to the wild-type. This result indicates that SmelC625 is regulated by ChvI, since its expression differed in the chvI mutants compared to wild-type, but other regulators may also be involved since a reciprocal change in SmelC625 expression was not observed in the chvI mutants compared to wild-type. Studying the S. meliloti ExoS/ChvI two-component pathway can provide greater insight into a symbiosis relevant to reducing greenhouse gasses and combating global warming. This research was supported by the CSU Special Fund for Research, Scholarship, and Creative Activity and National Institute of General Medical Sciences of the National Institutes of Health Award Number SC3GM144065 to E.J.C. | Poster #: 85 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: memory, sleep, synapse Project Title: Olfactory synapses are modulated by odor training and sleep in Caenorhabditis elegans. Author List: Garcia, Vanessa; Undergraduate, Biology, San José State University, Presenting Author
Odisho, Emma; Undergraduate, Biology, San José State University, Presenting Author
Soohoo, Emily; Graduate, Biology, San José State University
Tokalenko, Kateryna; Undergraduate, Biology, San José State University
Bykov, Andrew; Undergraduate, Biology, San José State University
Fung, Brandon; Undergraduate, Biology, San José State University
Guillen, Hazel; Undergraduate, Biology, San José State University
Harris, Malcolm; Undergraduate, Biology, San José State University
Jimenez, Vanessa; Staff, Biology, San José State University
VanHoven, Miri; Faculty, Biology, San José State University Abstract Sleep is essential for memory consolidation and can modulate synapses, but little is known about the effect of sleep on specific synapses. We hypothesize that sleep may be required to alter synapses in animals that retain long-term memory. Our goal is to investigate this hypothesis using the model organism C. elegans, which exhibits long-term olfactory memory after multiple cycles of odor training. C. elegans are innately attracted to butanone, an odorant secreted by some bacterial food sources. However, C. elegans can be trained to reduce this attraction if they are trained by exposure to butanone in the absence of food. After three training cycles, animals decrease their movement for approximately two hours, then retain the memory of reduced attraction to butanone for over 16 hours. We examined synapses between the butanone-sensing AWC olfactory neuron pair and their primary postsynaptic partners, the AIY interneurons, to determine if they were altered by training and quiescence. Synapses were visualized using the fluorescent split GFP-based trans-synaptic marker Neuroligin 1 GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP). We found that AWC-AIY NLG-1 GRASP synaptic intensities in butanone-trained animals were significantly reduced 16 hours after training if animals were allowed to be quiescent after training. However, animals trained with a control buffer or whose quiescence was disrupted mechanically did not display synaptic changes. Synaptic reductions in butanone-trained animals may explain their diminished response to butanone, and we propose that this synaptic remodeling may represent the memory trace. To understand whether post-training quiescence represents a sleep-like state, our group conducted pharyngeal pumping assays. Different forms of nematode sleep involve reduction and sometimes complete cessation of pharyngeal pumping. We found that butanone-trained animals had decreased pumping rates in the first two hours after butanone training. This is consistent with movement and posture data collected by our collaborators in the L’Etoile laboratory at UCSF, which indicate a sleep-like state after training. Our findings indicate that odor training and sleep collaborate to alter specific olfactory synapses in C. elegans. Defining the mechanisms by which sleep promotes memory in C. elegans may lead to the development of more effective treatments for diseases in which memory is affected, such as Alzheimer’s disease and dementia. | Poster #: 86 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: olfaction, synapse, neurscience Project Title: C. elegans exhibit postembryonic activity-dependent synapse formation Author List: Akitt, Sophia; Undergraduate, Biology, San José State University, Presenting Author
Aseoche, Darren; Undergraduate, Biology, San José State University
Byrd, Decklin; Undergraduate, Biology, San José State University, Presenting Author
Venkatesh, Roshna; Undergraduate, Biology, San José State University
Kaur, Sukhdeep; Staff, Biology, San José State University
Briseno, Fabiola; Staff, Biology, San José State University
Graves, Josiah; Staff, Biology, San José State University
Jimenez, Vanessa; Staff, Biology, San José State University
Nassif, Cibelle; Graduate, Biology, San José State University
VanHoven, Miri; Faculty, Biology, San José State University Abstract The nervous system is a complex network of neuronal cells that allow for communication throughout the body. In vertebrates, synapses are shaped by activity in the environment after birth, allowing individuals to learn and adapt to their environments. However, much is unknown about the mechanisms by which sensory activity impacts post-embryonic synapse formation. Understanding the molecular mechanisms that underlie sensory activity-dependent synapse formation may help to better treat neurological disorders. To investigate these mechanisms, we are studying the model organism Caenorhabditis elegans, which has a well-characterized and compact nervous system and is amenable to genetic manipulation. We utilize the transgenic trans-synaptic fluorescent marker Neuroligin-1 GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP) to visualize specific sensory synapses between PHB chemosensory neurons and AVA interneurons. We also utilize a PHB circuit-specific behavior assay to assess circuit functionality. Our group previously discovered a burst in PHB-AVA sensory synaptogenesis soon after hatching. However, this synaptic burst is reduced in genetic mutants with PHB neuron cilia defects that impair PHB sensory activity (che-3). Similarly, loss-of-function mutations in the PHB expressed G-protein coupled receptor SRB-6 have a reduced synaptic burst, consistent with sensory activity being required to promote synaptogenesis. Double-mutants homozygous for mutations in both che-3 and srb-6 are not significantly different from either single mutant, indicating that they likely act in the same sensory activity-dependent process. Expression of srb-6/GPCR in PHB neurons was sufficient to rescue the srb-6 loss-of-function mutant phenotype, consistent with the gene acting in PHB sensory neurons. In addition, overexpression of srb-6/GPCR in PHB was able to drive additional synaptogenesis, consistent with activity being sufficient to drive synaptogenesis. Our preliminary data indicate the neurite contact is not significantly disrupted in che-3 or srb-6 mutants, suggesting that disrupting sensory activity does not reduce synaptogenesis simply by impacting axon guidance. Our results indicate that like vertebrates, C. elegans have sensory-activity dependent synaptogenesis. Given the genetic tractability and imaging tools available, the discovery of activity-dependent synaptogenesis in C. elegans may allow us to better understand this crucial process at the molecular level. | Poster #: 87 Campus: California State University, Northridge Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: ethanol, drosophila, gene expression Project Title: Characterization of gene expression changes following repeated ethanol intoxications in the fruit fly Drosophila melanogaster Author List: Bonilla, Michelle ; Graduate, Biology, California State University, Northridge
Coreas, Jocelyn ; Graduate, Biology, California State University, Northridge
Massoud, Merna; Undergraduate, Biology, California State University, Northridge
Rodriguez, Cristian ; Undergraduate, Biology, California State University, Northridge
Abstract There has been a recent renewed interest in the transmission of acquired traits, particularly those related to tolerance to drugs and environmental toxins. Since tolerance is an accepted pre-requisite and strong predictor of addiction, a firmer understanding of the mechanisms underlying the transmission of tolerance could facilitate novel research avenues of importance in public health. Despite the significant advances made in characterizing the epigenetic and molecular mechanisms that underlie the transmission of acquired traits across diverse species, it is still unclear what genetic pathways may connect acquired traits that operate primarily at the metabolic, physiologic, or nervous system levels with the epigenetic modifications in germline that are necessary for heredity. While classic model organisms, such D. melanogaster and C. elegans are prime candidates to dissect conserved aspects of the genetic and epigenetic underpinnings of acquired trait inheritance, little is known about their capacity to transmit acquired drug tolerance to their progeny.
We have recently observed that parental flies that are intoxicated multiple times with ethanol vapors (once a day, for 10 minutes, over a 2 week period) give rise to progeny that is significantly more resistant to the sedative effects of ethanol and determined that such transmission of resistance is primarily matrilineal. Our findings present D. melanogaster as a suitable experimental model for forward and reverse genetics approaches to investigate the pathways connecting repeated exposures of an adult animal to ethanol and the necessary germline modifications that underlie the transmission of increased resistance in their progeny to the drug. We are now conducting RT-qPCR to determine if there are any differences in mRNA abundances of genes known to mediate ethanol tolerance and ethanol resistance in flies (Adh, Slo, Hang) from both ovaries from females repeatedly exposed to ethanol compared to those mock exposed to air, and brains of their respective progeny. Our findings will hopefully identify potential molecular mechanisms underlying the transmission of ethanol resistance, and thus create opportunities for future research. | Poster #: 88 Campus: California State University, Sacramento Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: CRISPR-Cas9, repair template, gene editing Project Title: Designing repair templates for CRISPR-Cas9 mediated knockout of nitrate reductase genes of the bacteria, Klebsiella oxytoca Author List: Aguilera, Marisa; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Olsan, Erin; Faculty, Biological Sciences, California State University, Sacramento Abstract Many members of the bacterial family Enterobacteriaceae are a normal part of the human gut microbiota. Members of this family include Salmonella enterica and Escherichia coli, which have been found to use anaerobic respiration using nitrate reductase enzymes to promote growth in low or no oxygen environments like the human gut. Another member of the family Enterobacteriaceae is the bacteria, Klebsiella oxytoca. Klebsiella oxytoca is a normal member of the human gut microbiota and can be an opportunistic pathogen. Recently, K. oxytoca was correlated with altered gut barrier function and dysbiosis (a disrupted and imbalanced gut microbiota) in a mouse model of cancer cachexia. The overgrowth of K. oxytoca in this model was proposed to be due to anaerobic respiration using nitrate. The goal of this study was to design single-stranded repair templates for CRISPR-Cas9 gene editing to knockout a nitrate reductase gene in K. oxytoca. Gene analysis was performed on K. oxytoca to identify several potential guide sequences and determine where the Cas9 protein will target the nitrate reductase gene, narI. Homology regions on each side of the double strand break were identified by determining where each double strand break will occur, selecting a 45-nucleotide sequence upstream of the guide sequence and one 45-nucleotide sequence downstream of the guide sequence. The two 45-nucleotide sequences were then combined to form a 90-nucleotide single stranded DNA repair template. This process was completed for multiple guide sequences. PCR (Polymerase Chain Reaction) primers for confirmation of the knockout were designed using the gene sequence that directly encompasses the guide RNA sequences and the two homology regions on each side of the break. These primers will be used to confirm where the double strand breaks are repaired, hopefully removing a portion of the targeted narI gene and rendering this gene non-functional. The knockout strains will be used to determine the necessity of narI to promote growth in an environment with low or no oxygen. | Poster #: 89 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: DNA barcoding, American pika Project Title: DNA barcoding of American Pika-associated plants: comparing the efficacy of two genetic markers, ITS and trnL, for plant identification Author List: Guru, Neha; Undergraduate, Biological Sciences, San José State University, Presenting Author
Martinez, Salvador; Undergraduate, Biological Sciences, San José State University, Presenting Author
Thang, Stephanie; Undergraduate, Biological Sciences, San José State University
Castillo Vardaro, Jessica; Faculty, Biological Sciences, San José State University Abstract American pikas (Ochotona princeps) are generalist herbivores, meaning they eat a wide variety of plants. However, they are known to preferentially consume some plant species over others. In our research lab, we are applying DNA metabarcoding to identify the plants within pika feces to characterize their diets. To determine whether pikas select certain plants and avoid others, we are comparing what we find in their feces to what we find within their territories and surrounding areas. In addition to collecting pika feces, we conducted vegetation surveys and collected plant samples. Many of these plants are challenging to identify in the field, so we are using a molecular identification approach to confirm field identifications and to generate a reference database to compare against the diet data. To accomplish this, we 1) extracted DNA from plants collected in northwestern Nevada and the Sierra Nevada mountains; 2) performed separate PCR reactions for two highly conserved genomic regions, the internal transcribed spacer (ITS) and the P6 loop of the chloroplast trnL intron; 3) sequenced the products using a high-throughput Illumina platform; and 4) compared the resulting sequences with the NCBI Genbank genetic sequence database. The trnL marker produces a significantly shorter fragment than ITS and therefore may be more successfully amplified from highly degraded DNA sources such as feces. This may be particularly true in cases such as American pikas where the fecal material is passed through the digestive system twice. However, shorter fragments may result in loss of taxonomic specificity, i.e., the ability to differentiate among closely related plant species. We found that ITS resulted in fewer matches overall and far fewer perfect matches with the public database, likely the result of a lack of representative sequences from our taxa of interest. In contrast, the trnL region often resulted in tens to hundreds of exact matches, indicating that it is not specific enough to differentiate among many of our plant taxa. As a result, we concluded that ITS is the more appropriate marker for our analyses. These results underscore the importance of appropriate marker choice, as well as collecting reference samples, when conducting diet analyses. | Poster #: 90 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: cardiomyocytes, adenovirus, imaging Project Title: Adenoviral-based tools to enable cardiomyocyte-specific imaging in heterogeneous primary culture using digital holographic microscopy Author List: Huang, Herman; Graduate, Biological Sciences, San José State University, Presenting Author
Gomez, Ayessa; Undergraduate, Biological Sciences, San José State University
Caampued, Andrew; Undergraduate, Biological Sciences, San José State University
Payumo, Alexander; Faculty, Biological Sciences, San José State University Abstract Adult mice and rats exhibit a limited degree of cardiac regenerative capability. However, neonatal rodents possess a robust capacity for heart regeneration, which depends on the proliferation of cardiomyocytes (CMs) - the muscle cells of the heart. The heart’s ability to regenerate is lost when neonatal CMs enter but fail to complete the cell cycle and become binucleated, a marker of permanent cell cycle arrest. While a number of physiological signals have been discovered to drive this process in vivo, the cellular mechanisms that inhibit the ability of CMs to divide are largely unknown. Our lab is developing a digital holographic imaging approach to visualize the direct effects of hormones on CM proliferative dynamics with three-dimensional single-cell resolution. Holographic microscopy creates phase images based on interference patterns generated between reference and sample beams. This enables “label-free” imaging of cultured cells. However, primary CMs isolated and cultured from newborn rat hearts are often a heterogeneous mixture of both CMs and non-CMs (e.g., fibroblasts, endothelial cells). The specific aim of this study is to develop adenoviral-based tools that will enhance our ability to specifically track CMs using our digital holographic imaging platform. Adenoviruses robustly infect CMs allowing expression of transgenes. Using a modular MultiSite Gateway cloning strategy, we have successfully assembled adenoviral expression constructs, transfected these constructs into AD293 cells, and generated adenoviruses that now allow us to test two independent approaches in parallel. In the first approach, we express a puromycin resistance gene downstream the CM-specific cardiac troponin T (cTnT) promoter. This tool will allow us to treat our cultures with puromycin, enrich for CMs, and facilitate CM-specific imaging using conventional digital holographic imaging microscopy. In a second approach, we express an enhanced green fluorescent protein (EGFP) reporter downstream of the cTnT promoter, which will permit selective fluorescent labeling of CMs when used in combination with next generation hybrid digital holographic and fluorescence imaging technology. Uncovering the cellular mechanisms that limit the ability of cardiomyocytes to successfully divide after birth will support new strategies in cardiac regenerative medicine to enable heart regeneration in adult humans. This work is supported by a R16GM146643 award from the National Institutes of Health.
| Poster #: 91 Campus: California State University, Los Angeles Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: MyoD, myogenesis, transcriptional regulation Project Title: How Do the MyoD Binding Regions Upstream and Downstream of the Acta1 Gene Influence Transcription of α-actin mRNA? Author List: Littlefield, Angela ; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author
DeSalvo, Gilberto, Ph.D.; Caltech
Sharp, Sandra B., Ph.D.; Faculty, Biological Sciences, California State University, Los Angeles
Abstract The transcription factor MyoD contributes to myogenesis (muscle development) by binding to conserved regulatory sequences typically found upstream of target genes. Interestingly, binding sites for MyoD have been found downstream as well as upstream of Acta1, one of MyoD’s target genes. The role of the downstream binding regions is unknown. I hypothesize that MyoD binding both upstream and downstream of Acta1 is necessary for developmentally regulated, muscle-specific Acta1 transcription. Testing this hypothesis will increase our knowledge of longer-distanced, downstream transcriptional regulation, and will provide regulatory mutations to test for in ACTA1-related myopathies that have not been ascribed to a coding-region mutation. I am performing transient plasmid transfections with normal and mutated Acta1 regulatory regions inserted upstream and downstream of a firefly luciferase reporter gene. The constructs are transfected into mouse pre-muscle and fibroblast cells, and a dual luciferase assay is performed at both myoblast (undifferentiated) and myocyte (differentiated) stages. I have determined the quantity of normalizer NanoLuc plasmid to use; have found that apparent NanoLuc activity is lower in growth versus differentiation conditions; and have observed an apparent effect of the specific plasmids present in the transfection mixture on NanoLuc activity. I address how these findings will inform our data analyses. I have performed a single trial transient transfection and luciferase assay on constructs containing the 5’, 5’ and various 3’, truncated 5’, and mutated 5’ binding sites. My results showed that normalized Acta1 promoter-driven firefly luciferase expression is higher in the presence of the upstream MyoD binding region than in its absence in myocytes, but not in myoblasts, consistent with previous reports. Additionally, constructs containing both the 5’ and 3’ binding sites, showed higher transcriptional activation when compared to constructs containing just the 5’ binding sites. So far, these results show that the 3’ binding site may play a role in increasing expression of the Acta1 mRNA. More trials with these constructs, as well as experiments testing additional constructs are underway.
(Funded by RISE NIH-R25 GM061331)
| Poster #: 92 Campus: California State University, Northridge Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: transcription factors, biodegradable plastic, starvation Project Title: Regulation of a dynamically-localizing polyester by three Caulobacter crescentus transcription factors Author List: Spinner, Tamryn; Graduate, Biology, California State University, Northridge, Presenting Author
Gonzalez, Erick; Undergraduate, Biology, California State University, Northridge, Presenting Author
Taylor, Eljoie; Staff, Biology, California State University, Northridge
Schreiner, Shannon; University of Southern California
Vu, Son; Olive View Hosptial
Arvizu, Ignacio; University of Southern California
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 activates the expression of the transcription factor PhaR, 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 (p)ppGpp, CtrA and PhaR stimulate 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. | Poster #: 93 Campus: California State University, Chico Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: toxicity, zebrafish, wastewater Project Title: Treated Wastewater Effluent Has No Negative Effect On Zebrafish Immune Development Author List: Rodriguez, Sofia; Graduate, Biological Sciences, California State University, Chico, Presenting Author
Stachura, David; Faculty, Biological Sciences, California State University, Chico Abstract Even though wastewater gets treated in sewage plants, there are still many ambiguous substances such as pharmaceuticals, insecticides, and chemicals in the final effluent (FE), the treated wastewater that gets discharged into local rivers. Environmental exposure to such substances have shown deleterious effects on the health of ecosystems and humans. We investigated what the effects of substances in FE had on the development and health of fish. To perform this study, Danio rerio (zebrafish) embryos were exposed to wastewater final effluent from Sacramento County to see if it negatively affected embryonic development. FE was collected and added to developing zebrafish at various dilutions at different time points during embryogenesis. Fish survival and morphology were examined and transgenic zebrafish with fluorescently labeled blood were used to determine the influence of FE on blood and immune cell formation. Comparing the survival, morphology, and blood formation in the control group to the treated group showed slightly lower survival, even though survival was over 85 percent across all groups. Morphological abnormalities and blood formation anomalies were observed in both control and treated groups; neither groups were significantly affected by exposure to FE. Overall, our results show that the FE is not detrimental to zebrafish immune development. The effect of FE on zebrafish helps us understand the risks of potential toxins present in treated wastewater and its possible effect on aquatic environments. This study highlights the importance of assessing the risks concerning how potential toxins present in FE can potentially affect the development of the immune system.
| Poster #: 94 Campus: California State University, Sacramento Poster Category: Other Keywords: gut-brain axis, Drosophila melanogaster, biomechanics Project Title: Kismet affects the gut-brain axis in Drosophila melanogaster Author List: Tran, Chau; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Eby, Wesley; Undergraduate, Physics and Astronomy, California State University, Sacramento, Presenting Author
Niosi, Angelo; Graduate, Biological Sciences, California State University, Sacramento
Sundar, Punithavathi; Graduate, Computer Science, San José State University
Vo, Nguyen (Henry); Undergraduate, Biological Sciences, California State University, Sacramento
Welch, Chloe; Graduate, Biological Sciences, California State University, Sacramento
Lee, Wendy; Faculty, Computer Science, San José State University
Jensen, Mikkel; Faculty, Physics and Astronomy, California State University, Sacramento
Morris, Eliza; Faculty, Physics and Astronomy, California State University, Sacramento
Mulligan, Kimberly; Faculty, Biological Sciences, California State University, Sacramento Abstract INTRODUCTION: The gut microbiome may contribute to the pathophysiology of neurodevelopmental disorders (NDDs), yet it is unclear how NDD risk genes affect gut physiology in a manner that may alter bacterial colonization. We addressed this question using Drosophila melanogaster with a mutation in kismet (kis), the ortholog of the human autism risk gene Chromodomain Helicase DNA Binding Protein 8 (CHD8). We used flies with heterozygous loss of kis to examine gut biomechanics, gut gene expression, gut microbiota, and the connection between gut microbiota and behavior.
METHODS & RESULTS: To quantify changes in gut tissue mechanics, we dissected whole guts from kis mutant and control flies and affixed them between two clips mounted on a high-precision force transducer and length controller, capable of measuring forces to micro-Newton precision. Our measurements revealed significant changes in the mechanics of kis mutant guts, in terms of elasticity, strain stiffening, and ultimate tensile strength. We used RNA-sequencing to investigate how gene expression was affected in kis mutant guts. Among the misexpressed genes were a subset involved in constructing the peritrophic matrix—an acellular matrix of glycoproteins and chitin that lines the insect gut lumen and can affect biomechanical properties. The peritrophic matrix also has a complex interplay with gut microbiota, so we next characterized the gut microbiome of mutants and controls by isolating genomic gut DNA and using 16S metagenomic sequencing. Analysis of the sequencing data showed that loss of kis profoundly reduced the abundance and diversity of microbiota in the gut, a hallmark of gut dysbiosis. To explore the connection between the gut microbiome and fruit fly behavior, we treated kis mutant and control flies with an antibiotic and then evaluated their courtship behavior. Depletion of the gut microbiome rescued courtship defects of kis mutant flies, indicating a connection between the mutant gut microbiome and behavior.
CONCLUSIONS: Our data shows that kis impacts gut mechanics, expression of gut genes affecting peritrophic matrix synthesis, and the gut microbiome. Further, manipulation of the gut microbiome affects Drosophila behavior. We propose that kis-mediated impacts to gut mechanics is a key component in the gut-brain interplay; future studies will address how antibiotic treatment affects gut mechanics to further study this phenomenon.
FUNDING: CSUS Research and Creative Activities Award.
| Poster #: 95 Campus: California State University, East Bay Poster Category: Other Keywords: acute pain, rat, inflammation Project Title: Effects of simultaneous inhibition of fatty acid amide hydrolase and soluble epoxide hydrolase on acute pain in male rats Author List: Yuan, Cassandra; Undergraduate, Psychology, California State University, East Bay, Presenting Author
Murray, Ashley; Undergraduate, Psychology, California State University, East Bay
Chin, Christopher; Undergraduate, Psychology, California State University, East Bay
Sanchez, Stephanie; Undergraduate, Psychology, California State University, East Bay
Fernandez, Alyssa; Undergraduate, Psychology, California State University, East Bay
Pecic, Stevan; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Kandasamy, Ram; Faculty, Psychology, California State University, East Bay
Abstract Current treatments for pain such as opioids and nonsteroidal anti-inflammatory drugs (NSAIDs) are limited in that they are only effective against mild pain and produce both dangerous and unpleasant side effects with repeated use. Further, new treatments for pain do not target novel mechanisms of action; thus, these drugs often have the same side effects as traditional treatments. Revolution in the treatment of pain requires identifying new drugs with novel mechanisms of action to afford pain relief without side effects. Fatty acid amide hydrolase (FAAH) and soluble epoxide hydrolase (sEH) are two enzymes that regulate pain and inflammation. Previous studies have demonstrated that co-administration of a FAAH inhibitor and a separate sEH inhibitor provides greater pain relief than administration of just one enzyme inhibitor. Based on these findings, we developed a novel dual inhibitor of FAAH and sEH, SP 4-5, that inhibits both enzymes simultaneously with just one drug. We hypothesized that simultaneous inhibition of FAAH and sEH using SP 4-5 will alleviate acute thermal and inflammatory pain in male rats. To test this hypothesis, we administered varying doses of SP 4-5 and evaluated their effects on three common pain tests in male rats. First, we used the tail flick test to determine whether SP 4-5 can produce relief against spinally-mediated pain. Administration of SP 4-5 and ketoprofen did not change the latency for the rat to flick its tail from a noxious heat source. Second, administration of SP 4-5 nor ketoprofen did not alter the latency for a rat to lick its paw when placed on a 52°C hot plate. Lastly, we injected dilute formalin into the hindpaw of the rat and observed licking and guarding behavior of the injected paw in two phases. The first phase is largely mediated by pain receptors and the second phase is mediated by inflammatory processes. Administration of SP 4-5 and the NSAID ketoprofen alleviated pain in the second, but not first, phase of the Formalin Test. Our results suggest that SP 4-5 alleviates acute inflammatory pain but does not affect acute thermal pain. Further, these results are consistent with the action of anti-inflammatory drugs indicating that SP 4-5 may also provide relief by altering inflammatory mechanisms. Future work will determine the dose- and time- relationship between SP 4-5 and other types of pain to better understand the therapeutic potential of dual FAAH/sEH inhibitors for the long-term treatment of pain. | Poster #: 96 Campus: San Diego State University Poster Category: Other Keywords: Heritability, Geometric morphometrics, Plasticity Project Title: Heritability and Phenotypic Plasticity of Body Size in the Parthenogenetic Wasp Dinocampus coccinellae Author List: Monahan, Scott; Undergraduate, Biology, San Diego State University, Presenting Author
Kristan, Adrian; Undergraduate, Biology, San Diego State University, Presenting Author
Sethuraman, Arun; Faculty, Biology, San Diego State University
Abstract Dinocampus coccinellae is a generalist Braconid parasitoid in the Euphorinae subfamily that reproduces through thelytokus parthenogenesis, a form of asexual reproduction where clonal diploid daughters emerge from unfertilized eggs. These unfertilized eggs are oviposited within a wide range of host coccinellid beetles, providing a variety of environmental conditions for the larval D. coccinellae to develop under. The clonal nature of D. coccinellae and the variety of environmental conditions available offers the opportunity to isolate environmental and genetic factors of development and quantify heritability and phenotypic plasticity in body size morphology. To accomplish this, multiple generations of unilineal (=reared on same host species) and multilineal (=reared on different host species) strains of D. coccinellae were established to isolate influences on larval development. With environmental and genetic factors held constant in the unilineal strain, a positive correlation between mother and daughter morphometrics would indicate strong heritability and variation can be quantified as the result of trait plasticity. With changing environmental conditions in the multilineal strain, a negative correlation between mother and daughter morphometrics would suggest weak heritability and a strong influence on larval development from host size morphology. Specimens from each generation were preserved and measurements were taken of various characteristics. A series of linear regressions were performed between mother and daughter wasps to test direct morphological relationships. Unexpectedly, our analyses determined (1) a negative correlation between size morphometrics of clonal mothers and daughters when reared on the same environment (unilineal), and (2) a positive correlation in parent-offspring body sizes when reared in different environments (multilineal). These results indicate that rather than environmental conditions or phenotypic plasticity being the sole determiners of offspring morphology, parental decisions in the tradeoff between fecundity and viability of offspring may have a stronger influence. | Poster #: 97 Campus: California State University, Chico Poster Category: Other Keywords: Eating disorder, Adolescence, Anxiety Project Title: Binge-eating alters anxiety-like behaviors in late adolescent female mice Author List: Rezaie Boroon, Parnaz; Graduate, Psychology, California State University, Chico, Presenting Author
Bates, Shawn; Faculty, Psychology, California State University, Chico Abstract Despite the increase in adolescents suffering from binge eating disorder, this phenomenon is still widely unexplored in scientific research. Furthermore, females are more likely to develop binge-eating disorder than males. As adolescent brains are not fully developed, commonly observed behaviors such as problematic eating may play a harmful role in the way their brain properly processes reward. This is especially important considering such alterations can render the adolescent brain more vulnerable to anxiety and depression-like behaviors. This study uses a novel model of binge-like eating behavior in adolescent mice to identify the ramifications of binge-eating disorder. Female adolescent C57BL/6J mice were exposed to standard chow or high-fat diet (HFD) for 2-h a day, three days a week (PND 42-72), for four weeks. Twenty-four hours later, cocaine reward were assessed. We also examined anxiety-like behavior using elevated plus maze (EPM), and depression-like behavior using the forced swim test (FST) and splash test. Animals exposed to HFD demonstrated greater food consumption, indicative of binge eating, during the four-week intermittent cycle compared to the standard chow group. The HFD group spent more time in the open arms compared to mice given standard chow during EPM. While HFD mice were slightly less immobile in FST compared to standard chow mice, HFD mice also groomed themselves noticeably less than standard chow mice during the splash test, suggesting an increase in depression-like behavior. Lastly, only the standard chow group developed preference for the compartment paired with cocaine. Our research findings suggest that binge eating in adolescence may contribute to increased anxiety- and depression-like behaviors in this age group. Future studies should explore the neural mechanisms underlying these behaviors. | Poster #: 98 Campus: San Francisco State University Poster Category: Other Keywords: electron microscopy, skate retina, neuroscience Project Title: The anatomy of rod pathways in the pure-rod retina of L. erinacea Author List: Umbertus, David; Graduate, Biology, San Francisco State University, Presenting Author
Anastassov, Ivan; Faculty, Biology, San Francisco State University Abstract The retina is a thin layer of tissue that lines the back of the eye and allows vertebrates to visualize the world around them. Most retinas use a duplex system of light sensitive cells known as rod and cone photoreceptors. Cones are used for photopic vision (daylight/color) and rods are used for scotopic vision (dim light). In most vertebrate retinas, rods utilize three distinct pathways for the transmission of visual information at different levels of brightness, called the primary, secondary, and tertiary pathways. The primary, secondary and tertiary pathways vary in sensitivity to light, with the primary being the most sensitive, and the tertiary the least sensitive. Interestingly, these pathways always utilize some form of cone circuitry, at the very least in mammals. The little skate (L. erinacea) is a type of cartilaginous fish with a pure-rod, “simplex” retinal system (that is, cones are entirely absent). Still, the skate retina can function under both dim and bright light conditions. Therefore, we are interested in how this retina manages to transmit information from rods to downstream bipolar and ganglion cells without the ability to piggy-back onto cone circuitry. We address this question by examining the anatomy of the little skate retina via 3D electron microscopy and immuno-staining. Previous data from our group suggests a hybrid rod-cone morphology in skate rods and we therefore hypothesize there might be hybrid morphology in the rod bipolar cell (the immediate connecting partner to rods), which also allows for direct connections from photoreceptors to ganglion cells (i.e., the cells responsible for retinal output). With this information, we can understand how the skate retina has evolved structurally to accommodate for the absence of cone photoreceptors and allow the skate to perform tasks in dim and bright light vision. | Poster #: 99 Campus: San José State University Poster Category: Other Keywords: Infection dynamics, Novel bacteriophage, Genome sequencing Project Title: Determining characteristics of a novel bacteriophage and applications to combat antimicrobial resistance Author List: Balitactac, Akiko Kaitlin; Undergraduate, Biological Sciences, San José State University, Presenting Author
Hayek, Edward; Undergraduate, Biological Sciences, San José State University
Cao, Karen; Undergraduate, Biological Sciences, San José State University
Lee, Wendy; Faculty, Computer Science, San José State University
Fowler, Robert; Faculty, Biological Sciences, San José State University
White, Steven; Faculty, Biological Sciences, San José State University
Singhal, Sonia; Faculty, Biological Sciences, San José State University
Abstract More than 2.8 million antimicrobial-resistant infections and 35,000 deaths occur in the United States every year. Bacteriophages (or “phages,” viruses that infect bacteria) are a good alternative to antibiotic treatments: Phages infect and kill antimicrobial-resistant bacteria, they target only specific types of bacterial cells, and they are non-toxic to human cells. We isolated a novel bacteriophage from Santa Clara County wastewater. We named the phage “Halophage'' due to its halo-shaped plaque appearance when grown on Escherichia coli B. Here we characterized the Halophage for future medical use. First, we extracted viral DNA and assembled the genome using Nanopore and Illumina sequencing. Second, we determined its course of infection on E. coli B using a one-step assay. We combined the phage with a liquid culture of E. coli B and allowed it to grow at 37 C. Every five minutes, we sampled from the culture onto LBGC agar plates with fresh E. coli B. The concentration of Halophage at each time point was estimated from the number of plaques on the plates. Third, we tested the Halophage’s host range, its ability to grow on other bacterial hosts. The halophage was combined with a sample of each host, plated on LBGC agar, and checked for plaque formation. Selected hosts were closely related to E. coli B or found within the gut microbiome.
The Halophage’s genome has 39,721 bp with 48.47% GC content and 62 predicted open reading frames. It shares 92% of its genome with the laboratory Enterobacter T7 phage. Initial one-step results indicate that its burst time (time from attachment to lysis of the host cell) is 28.75 minutes, and each infection releases an average of 252.7 new phages. It cannot infect Citrobacter freundii, Enterobacter cloacae, or Enterobacter aerogenes. We will follow up with host range tests of other bacteria found in the gut microbiome, such as E. coli K12, Lactococcus lactis, and Clostridium sporogenes.
Results from these experiments will pave the way for alternative treatment of antimicrobial resistant infections. The Halophage’s host range will provide information about bacterial infections besides those caused by E. coli B the phage has potential to cure. The one-step assay can be used to establish best treatment practices, such as methods of administering the phage and frequency of doses. With the genome, the genetic underpinnings of these characteristics can be determined to engineer the phage for optical medical use. | Poster #: 100 Campus: California State University, San Bernardino Poster Category: Other Keywords: rats, sensitization, associative learning Project Title: Role of environmental conditioning in the cross-sensitization between RU 24969 and cocaine in juvenile rats. Author List: Cotter, Laura ; Undergraduate, Psychology, California State University, San Bernardino, Presenting Author
Robinson, Jasmine; Graduate, Psychology, California State University, San Bernardino, Presenting Author
Lujan, Alejandra; Undergraduate, Psychology, California State University, San Bernardino
Uribe, Paolo; Undergraduate, Psychology, California State University, San Bernardino Abstract Recently, we found that cocaine and the nonselective 5-HT1A/1B receptor agonist RU 24969 show reciprocal cross-sensitization using a multi-trial procedure in preweanling rats. Interestingly, when a one-trial pretreatment regimen was used, cross-sensitization was only apparent when preweanling rats were pretreated with RU 24969 and tested with cocaine, but not the reverse. Since one-trial sensitization is known to be more context dependent than multi-trial procedures, it is possible that the discrepancies in cross-sensitization may be caused by differences in associative learning. Thus, we assessed the role of environmental cues by altering the drug pretreatment context. Specifically, on the pretreatment day [postnatal day (PD) 20], rats were injected with saline, cocaine (30 mg/kg), or RU 24969 (5 mg/kg) and placed in one of three pretreatment compartments (activity chamber, operant chamber, or home cage) for 45 min. Thirty min after being returned to the home cage, rats given cocaine or RU 24969 were given an injection of saline. Rats that had received saline initially were divided into three groups and received a saline, cocaine, or RU 24969 injection. Two days later (PD 22), rats that received cocaine during pretreatment were challenged with RU 24969 (5 mg/kg) and rats that received RU 24969 were challenged with cocaine (20 mg/kg). Rats that had previously received two saline injections received either cocaine or RU 24969. All rats were placed in the activity chamber after injection for a 120 min. Thus, rats in this experiment were either pretreated in the same activity chamber as they were tested (associative group), pretreated in their home cage and tested in the activity chamber (non-associative familiar group) or pretreated in the operant chamber and tested in the activity chamber (non-associative novel group). Like our earlier finding, cross-sensitization was only seen in rats pretreated with RU 24969 and challenged with cocaine. This cross-sensitization was seen in both the associative and non-associative novel groups but not in the group that received their drug in the home cage. Unexpectedly, we found rats pretreated with cocaine showed a decreased response to RU 24969 that was strongest in the non-associative familiar group. In summary, these results indicate that context is important in the cross-sensitization of cocaine and RU 24969, but it is unclear if associative learning is mediating the effect of these environmental cues. |
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