2012 Presentations

ACS 2012

Synthesis of Polyols From Seed Oils With Various Applications From Surfboards to Drug Delivery Systems

Ayalew, Luladey Tuey, Stacey M., Dr. Michael F. Z. Page
Chemistry Department, California State Polytechnic University of Pomona

With petroleum reserves becoming limited vegetable oils have been reported as a sustainable alternative in the synthesis of polymers. In this study, polyols were synthesized through the epoxidation and hydroxylation of sunflower seed oil and linseed oil. The epoxidation intermediate was confirmed by analyzing the respective spectra and detecting a new resonance at 822.2 cm-1 (FT-IR) and a multiplet at δ = 2.9-3.1ppm (1H NMR). Once hydroxylated, the polyols can be utilized to produce a variety of materials of interest in our lab that include a nano-particle drug delivery system and an eco-friendly surfboard. These synthetic advances will lead to the inclusion of seed oils in consumer products and biomedical medical research.


Phylogenetic Placement of the Enigmatic Opisthobranch Genera Doridoxa and Bathydoris within Nudibranchia

J. Mahguib, Angel A. Valdes
Biological Sciences Department, California State Polytechnic University, Pomona, CA

Introduction: Opisthobranch sea slugs are a highly diverse group of aquatic gastropod mollusks found in nearly all marine ecosystems. Nudibranchia is the most diverse clade of opisthobranchs and contains some of the most species rich groups, such as the dorids (Doridoidea) and the cladobranchs (Cladobranchia). The opisthobranch genera Doridoxa and Bathydoris have in the past been placed at varying positions along the nudibranch phylogeny. Odhner placed them as sister to each other within a group called Gnathodoridacea, which was sister to the dorids. Tardy placed Bathydoris as sister to the dorids within a group called Euctenidiacea, and placed Doridoxa as sister to Euctenidiacea. Schrödl, Wägele and Willan placed Doridoxa as sister to the cladobranchs. All these hypotheses were based on morphological characters alone, due in part to the unavailability of DNA sequencing technology (for older studies) and to a lack of readily available specimens from either group. For the present study I had access to representative specimens, which have thus far yielded clean, usable mitochondrial gene sequences (CO1 and 16S). Nuclear genes (H3 and 18S) will be sought after for this project as well. The goal of this study is to use gene sequences to build a phylogeny of nudibranchs to resolve the long-standing controversy surrounding the phylogenetic placement of Doridoxa and Bathydoris.

Methods: DNA was extracted from sample specimens using various extraction protocols including Chelex, DNeasy, and CTAB. Specific genes of interest were targeted using stock primers and amplification was done via polymersase chain reaction (PCR) using an eppendorf Mastercycler personal and a TECHNE TC-3000X thermocycler. Confirmation of gene amplification was done through agarose gel electrophoresis. Purification of successfully amplified gene products was done using the Montage purification kit. Nucleotide concentrations were obtained using a Thermo Scientific NanoDrop 1000 spectrophotometer. DNA sequencing was done at City of Hope in Duarte, CA. Sequencing results were downloaded and imported into Geneious for assembly, cleaning, extraction of consensus, alignment and concatenation of sequences. A Bayesian analysis using concatenated CO1 and 16S sequences from representative specimens of all the major groups in Doridoidea and Cladobranchia was conducted to reconstruct the phylogeny of the Nudibranchia.

Results: Analysis of our sequencing data revealed that the genus Doridoxa is embedded within the Cladobranchia. Bathydoris was revealed to be a sister group to the clade formed by the cladobranchs plus Doridoxa

Conclusions: These findings support the placement proposed by Schrödl, Wägele and Willan whereDoridoxa is concerned, and offer a novel placement for Bathydoris within Nudibranchia. The data supports Doridoxa as being a more derived group of nudibranchs than previously considered.

Two Cryptic Sympatric Species of Costasiella in The Bahamas Evolved Allopatrically

Erika R. Espinoza, and Ángel Valdés
Department of Biological Sciences, California State Polytechnic University, 3801 West Temple Avenue, Pomona, California 91768, USA

Costasiella ocellifera, Simroth 1895, is a species of sea slug (Mollusca: Gastropoda; Sacoglossa) that is found throughout the Caribbean. This species feeds exclusively on Avrainvillea green algae upon which is highly camouflaged. A preliminary phylogenetic analysis was constructed from nuclear (H3) and mitochondrial (16S, COI) gene sequences. It shows that several specimens identified as being C.ocellifera and collected from the Bahamas are genetically distinct from other specimens of C. ocelliferafrom the Bahamas and other western Atlantic locations. These genetically different specimens are considered to belong to a cryptic, undescribed species. The new species looks very similar (almost identical) to the typical C. ocellifera but a few differences in coloration and radular morphology provide additional support for molecular results.
Costasiella ocellifera and the new species are sister and sympatric in the Bahamas. Sister species with overlapping ranges, inhabiting areas in which there is no evidence of present or past biogeographic barriers to dispersal, could constitute potential cases of sympatric speciation. In this case, however, both species feed upon the same species of Avrainvillea and were collected in the same locations during the same time of the year. This seems to reject any possibility niche partition or allochrony, which are common pre-requisites for sympatric speciation. Thus, we hypothesize that these two species evolved allopatrically. Lack of evidence of hybridization (no nuclear alleles of the new species are found in C. ocellifera or vice versa) suggests reinforcement.
This case is similar to other reported new cryptic species in the Bahamas (Chelidonura, Philinopsis, Spurilla) and provides further evidence of a partial or complete interruption of gene flow between the Bahamas and other Caribbean areas at some point in the past.

Study of autolysins and autolysis patterns of Clostridium botulinum

Jessica Jackson and Wei-Jen Lin
Biological Sciences Department, California State Polytechnic University, Pomona

Introduction: Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium that produces the most potent toxin known to man, the botulinum neurotoxin (BoNT). BoNT causes botulism, a neuroparalytic disease found in humans and animals. The toxin poses severe health risks to humans, and is a major concern for public health officials. Our previous studies show that the toxin is produced during cell growth and released as early as mid-log phase of bacterial growth. We believe autolysins, which hydrolyze the cell wall, are responsible for the cell leakage and early release of the toxin. In this study we analyzed the autolysins of C. botulinum and the different autolysis patterns of three type A C. botulinum strains

Methods: Autolysis was studied in three C. botulinum strains by monitoring optical density throughout the growth curves to compare the autolysis of these strains grown in two different media. Another method of studying autolysis used fluorescent dyes to determine cell membrane permeability. Bioinformatics research was done comparing C. botulinum type A strain ATCC3502 to Bacillus subtilisstrain 168, a model strain with characterized autolysin functions. BLAST was performed on the protein sequences of B. subtilis compared to C. botulinum. Transglycosylase CBO 3012 was further analyzed for its phylogenetic comparison to other species using ClustalW. 

Results: Our results show that autolysis occurred in all three strains of C. botulinum, and the rate of autolysis varied due to the strains and growth media. The BLAST results of the genomic sequence analysis showed over 100 C. botulinum genes with homologies to B. subtilis str. 168. Further analysis of CBO 3012, a transglycosylase, showed greater than 98% identity and homology to other C. botulinumtype A strains. Different C. botulinum serotypes showed greater than 53% identity and 73% homology to CBO 3012. 

Conclusions: Phylogenetic analysis of the transglycosylase CBO 3012 showed the evolutionary relationship of this autolysin in a variety of bacteria, which could lead to a better understanding of the function of this gene, as well as this enzyme’s target site. The expression of the autolysin genes will be further analyzed using microarray analysis of the bacterium grown in the two media that exhibited different autolysis patterns. This data could possibly help correlate autolysins being responsible for bacterial cell lysis and early release of the toxin. The protein sequence alignment and microarray analysis will bring us one step closer to understanding the functions of autolysins in C. botulinum. A better understanding of the mechanism of toxin release and the role of autolysins may lead to better treatment options against this bacteria and its neurotoxin.

SCCUR 2012

Investigation of the Roles of RNA-binding Proteins in Arabidopsis thaliana

Aubrie De La Cruz, California State Polytechnic University Pomona, Maureen Hummel, University of California and Dr. Julia Bailey-Serres, Professor of Genetics, University of California Riverside

Abstract: There are very few existing investigations of the functions of RNA-binding proteins (RBPs) inArabidopsis thaliana. Over 1200 RBP domains have been identified in the organism, but only one-third of their functions have been characterized. These compounds play major roles in post-transcriptional RNA modification, and it is crucial to explore how these roles might affect stress responses inArabidopsis. To determine the localization of two of these proteins (RBPa and RBPb) within the cell,Arabidopsis Col-0 (WT) protoplasts were transfected with vectors that contained an RBP-mCherry construct and then viewed using a confocal microscope to detect fluorescence within the cell. To see how rbp mutants responded to different stresses, mutant lines rbpa and rbpb, and Col-0 were grown on several different media including 300 mM sucrose, 2 µM Abscisic acid (ABA), 2 µM Paclobutrazol (PAC), 300 mM sorbitol and 0.01% DMSO. Mutant seedlings were then monitored for differences in % germination, % greening, and root length compared to Col-0. To determine how abundantly RBPb was expressed in Col-0, rbpb-1 and rbpb-2 cells, immunopurification (IP) and Western Blotting were conducted using a specific α-RBPb antibody. Results of all these studies showed that both RBPa and RBPb localized in the cytoplasm. When grown on PAC for 8 days, rbpa mutants had a lower % germination and lower % greening, compared to Col-0. When grown on PAC for 7 days, rbpb-2 mutants maintained a high % germination and % greening in comparison to Col-0. When grown on a high sucrose level for 14 days, rbpa mutants exhibited shorter roots compared to Col-0. The Western blot showed that α-RBPb could likely detect RBPb. Future experiments will focus on improving the RBPb immunopurification, and examining possible non-cytoplasmic localization of the RBPs in Arabidopsis.

ASM 2012

The Effects of Estrogen on the Susceptibility to Candida Albicans Infection in c57BL/6 Mice

Melissa Arroyo-Mendoza, Nancy E. Buckley
Department of Biological Sciences, California State Polytechnic University, Pomona, CA

Candida albicans (C. albicans) is the most common fungal pathogen affecting immune compromised individuals. Recently, in our laboratory, we found that immunocompetent female c57BL/6 mice are more resistant to a systemic C. albicans infection than male mice. In addition to being the main sex hormone in females, estrogen is also able to regulate thymic development and immune function. This is due to estrogen receptors found not only in reproductive tissue, but also on certain immune cells including monocytes, macrophages, and T cells. Therefore, we proceeded to investigate the role of estrogen in the resistance to this systemic yeast infection.  Thus female, male or castrated male c57BL/6 mice (n=4-7) were used.  The mice were implanted (SC) with placebo or estrogen (0.09-1.4mg/pellets) pellets.  Seven days after pellet implantation, mice were infected with 5.5 x 105 C. albicans/mouse (i.v.) and observed daily for morbidity and survival for up to thirteen days. To determine tissue fungal load, serum estrogen levels and cytokine levels, some mice were sacrificed five days after yeast infection. The tissue fungal load was assessed as yeast colony forming units/g tissue (CFU/g). The serum collected was analyzed by estrogen or cytokine specific Enzyme-linked immunosorbent assays.  We found that estrogen supplementation did not improve survival of castrated males, it fact, estrogen supplementation worsened survival, tissue fungal load and serum IL-6 levels. Interestingly, castrated males receiving placebo had similar survival rates and fungal load as those of females.  However, serum IL-6 levels in castrated males receiving placebo were slightly elevated compared to those in females. Our findings show that estrogen alone is not protective of the C. albicans systemic infection outcome in male mice and suggests that other factors are involved.  A broader study of the cytokine profile may elucidate the role of pro-inflammatory cytokines in the disease course in males compared to females.

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