Long term protection in Swiss Webster(SW) mice using a liposomal M2e Influenza A vaccine
J. E. Henriquez, N. Nguyen, E. Chavez, Jill Adler-Moore
Cal Poly Pomona
Long term protection in Swiss Webster(SW) mice using a liposomal M2e Influenza A vaccine
J. E. Henriquez, N. Nguyen, E. Chavez, Jill Adler-Moore
Cal Poly Pomona
Introduction: Liposomal M2e (L-M2e) Influenza A vaccine was shown in our laboratory to provide significant protection against Influenza A challenge one week post-vaccination in SW mice. The present study focused on how long the protection generated by the vaccine would last, with or without an additional boost.
Methods: Four gps (A-D, n=21/gp) of SW mice were vaccinated with L-M2e (100ug M2e/dose) or non-M2e liposome control (Con) using a Subcutaneous (SC) prime on day 0 and intranasal (IN) boosts on d28 and d56. Mice in gps A (M2e) and B(Con) were given an additional IN boost 4 weeks after completing the standard vaccination regimen, while gps C (M2e) and D(Con) had no extra IN boost. Four days after the gp A boost, serum and spleens from 6 mice/gp were collected and remaining mice in each gp (n=15) challenged with 10X LD50 of influenza A PR8 H1N1. Day 5 post-infection, lungs from 5 mice/gp were collected for viral burden analysis using a foci assay, and the rest of the mice (n=10) were followed for morbidity. Spleens were used for BioPlex cytokine secretion and EliSpot assays; serum was used for determining viral precipitating antibody titers and anti-M2e IgG isotypes by ELISA.
Results: Vaccinated mice given the extra boost of L-M2e (gp A) showed significantly less weight loss (P<0.0001), and reduced disease signs (P<0.01) as well as prolonged survival (90%) versus non-boosted L-M2e mice (50% survival) (gp C). Viral lung burden was also reduced in boosted mice compared to their Con (P<0.02). However, both boosted and non-boosted mice had significantly prolonged survival versus their respective Con (0% survival) (P<0.0004 and P<0.019, respectively) and IFN-y levels were increased for both gps versus their Con (P<0.02). Boosted mice had significantly higher precipitating antiviral antibody titers compared to their Con(P<0.009) while higher concentrations of anti-M2e IgG1 and IgG2A were detected in boosted and non-boosted mice versus their respective Con(P<0.001).
Conclusion: Long term protection in Swiss Webster mice against lethal influenza virus infection could be achieved by L-M2e vaccination, with or without an extra IN boost. However, the additional IN boost provided an enhanced level of protection since the mice given the extra boost had a higher survival rate with reduced infection severity compared to non-boosted mice. Acknowledgements: NIH MBRS RISE 2 R25 GM061190-05A2, Molecular Express Inc. Key Words: influenza, vaccination, liposomes
Fast Events Revealed by Concentration Jumps in ℽ-Aminobutyric Acid Transporters
Rachel V. Sanchez, Cynthia M. Anderson, Sepehr Eskandari
Biological Sciences Department, California State Polytechnic University, Pomona
ℽ-Aminobutyric acid (GABA) transporters (GAT) are electrogenic transport proteins that couple the cotranslocation of Na+, Cl–, and GABA across the plasma membrane of neurons and glia. We used rapid extracellular Na+ or Cl– concentration jumps over intact, voltage-clamped Xenopus laevis oocytes expressing GAT1 to gain insight into the nature of partial reactions responsible for ion binding to the transporter. In the presence or absence of external Cl–, Na+ concentration jumps yielded capacitive charge movements that were characteristic of positive charge entering/leaving the membrane electric field. Sodium removal led to an outward capacitive current, and subsequent reintroduction of Na+ led to an inward capacitive current of equal magnitude. The charge movements were characterized by a rapid transient rising phase followed by two-component exponential decay to a steady-state. The steady-state component was also seen in control cells and was subtracted from the total current to yield the transporter-mediated charge movement. The charge movements evoked by Na+ concentration jumps were voltage-dependent, saturating at membrane potentials more negative than –90 mV, but decreased sharply at more positive potentials. In the nominal absence of external Cl–, there was a ~70% reduction in the total charge moved in response to Na+ concentration jumps. The time-to-peak for the rising phase, characteristics of the two components, and the time constants of the relaxation of the charge movements were not altered significantly in the absence of external Cl–. Chloride concentration jumps in the presence of Na+ exhibited charge movements that were qualitatively similar to those observed with Na+ jumps. Rapid Cl– removal led to an outward capacitive current and Cl– reintroduction led to an inward capacitive current. At any given voltage, the total charge moved in response to Cl– jumps was ~30% of the charge moved with Na+ jumps in the presence of Cl–. In the absence of Na+, Cl– did not evoke any charge movements. A specific inhibitor of GAT1, SKF-89976A, also led to charge movements. Altogether, the results suggest that external Cl– does not play a significant role in Na+ binding to the transporter, but rather it stabilizes the fully Na+-loaded transporter state. We propose Na+ binding constitutes the rate-limiting step in the GAT transport cycle, whereas GABA binding and translocation across the plasma membrane are rapid events. Supported by NIH grant SC1GM086344.
Keywords: neurotransmitter transporter, concentration jump, electrophysiology
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 where Doridoxa 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. ocellifera from 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 subtilis strain 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. botulinum type 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.
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) in Arabidopsis 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 in Arabidopsis. 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.
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.
Comparison and analysis of Clostridium botulinum autolysins
Jessica Jackson, Jade Hardy, and Wei-Jen Lin
Department of Biological Sciences, California State Polytechnic University, Pomona
Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium. It produces botulinum neurotoxin (BoNT), a potent toxin responsible for botulism in humans and animals. BoNT poses severe health risks to humans, and is a major concern for public health. Previous studies in our lab have shown 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. Better understanding of the toxin and how it is released could lead to treatment and prevention of botulism. In this study we analyzed the autolysins of C. botulinum using microarray and genomic sequence analysis. Bioinformatics analysis was done comparing C. botulinum type A strain ATCC3502 to Bacillus subtilis strain 168, a model strain with characterized autolysins. BLAST was performed on the protein sequences of B. subtilis compared to C. botulinum. Transglycosylase CBO 3012 was further analyzed for its expression pattern by microarray and for its phylogenetic comparison to other species using ClustalW. The BLAST results of the genomic sequence analysis showed over 100 C. botulinum genes with similarities and homologies to B. subtilis str. 168 autolysins. Further analysis of CBO 3012, a transglycosylase, showed greater than 98% identity and homology to other C. botulinum type A strains. Different C. botulinum serotypes showed greater than 53% identity and 73% homology, and other Clostridium species showed between 52-96% identity and 70-99% homology to CBO 3012. There were less than 57 and 77% identity and homology, respectively, for other bacterial genera compared to CBO 3012. Phylogenetic analysis of 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 its evolutionary path. Microarray expression analysis of C. botulinum ATCC3502 reveals autolysin activity in early mid-log phase of bacterial growth. Microarray data confirms that several autolysins are active around the same time the toxin is being released, possibly correlating autolysins being responsible for early cell lysis and release of the toxin. Identities and homologies shown between C. botulinum ATCC3502 and B. subtilis str. 168 brings us one step closer to defining the precise functions of autolysins in C. botulinum.
The Effect of Nectar levels on Honey Bee (Apis mellifera) Visit Duration
and Pollen position in Watermelon (Citrullus lanatus)
Accurate estimates of pollinator efficiency of bee species are essential to optimizing commercial crop pollination. One common method of estimating pollinator efficiency is to quantify the number of pollen grains deposited from a single visit to a previously unvisited flower. The amount of pollen deposited by this first visit is presumed similar to the amount of pollen deposited per visit for all visits a flower receives from a given kind of pollinator. In watermelon, floral nectar levels are higher initially and then decrease throughout the day. If higher nectar levels cause longer visits and greater pollen deposition, then per visit pollen deposition is likely to vary temporally and cannot be approximated by pollen deposition from the first visit a flower receives. This study investigated the effect varying floral nectar levels have on visit duration and pollen deposition by honey bees. Nectar from virgin flowers was removed with microcapillary tubes (with full extraction, 1 μL extraction and no extraction control treatments) and offered to honey bees for a single visit. The nectar left in full extraction flowers was similar to levels in open pollinated flowers. Each visit duration was recorded. Pollen deposited was counted. Significant differences were identified using ad hoc Tukey-Kramer tests. Flowers from 1 μL extraction and control treatments received longer visits than full extraction flowers (p=.0121 and p=.0002, respectively). Control and 1 μL extraction flowers had more pollen deposited on than full extraction flowers in terms of stigmatic pollen (p=.0016, p=.0039) and total pollen (p=.0033, p=.0246). Mean total pollen deposition by honey bees was 2.7 times greater on control flowers than full extraction flowers. Our data indicate that honey bee visit duration and per visit pollen deposition is strongly influenced by nectar levels in watermelon, which has important consequences for understanding and measuring honey bee pollinator efficiency.
Functional Consequences of Sulfhydryl Modification of the g-Aminobutyric Acid Transporter 1 (GAT1) at a Single Solvent-Exposed Cysteine Residue
Rachel Sanchez, Jaison Omoto, Ali Rahnama-Vaghef, Matthew Maestas Cynthia Anderson, Esther Choi, Gerardo Salto, and Sepehr Eskandari
γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter of the central nervous system (CNS). GABA molecules act on GABA receptors on the postsynaptic neuron to induce an inhibitory postsynaptic response, thus reducing excitatory transmission. GABA concentrations in the brain extracellular fluid must be maintained at low resting levels to ensure a proper balance of excitatory-inhibitory response in the CNS. GABA transporters located on the presynaptic neuron and surrounding glia regulate inhibitory neurotransmission by removing GABA molecules from synaptic and extra-synaptic regions. GABA transporters are electrogenic cotransporter that utilizes the Na+ electrochemical gradient to translocate GABA molecules across the plasma membrane and into the presynaptic neuron and glial cells. Each translocation cycle, transports 2 Na+ : 1 Cl−: 1 GABA into the cell.
The focus of this study was to label the cysteine residues of GABA transporter 1 (GAT1) facing the extracellular milieu in order to gain an understanding of the functional consequences on steady-state kinetics resulting from the labeled residues. Three of the fourteen cysteine residues are predicted to be facing the extracellular fluid. Residue C74 is positioned at the membrane/extracellular interface of transmembrane domain one and is thought to contain the only exposed free thiol group. Cysteine residues C163 and C174 are located in the extracellular loop, which links transmembrane domains three and four. Under most conditions, it is suspected that C163 and C174 interact to form a disulfide bridge. Human GAT1 was expressed in Xenopus laevis oocytes by injecting the cells with human GAT1 cRNA. Electrophysiological methods were performed using a two-microelectrode voltage clamp method in order to assay for transporter function before and after labeling. GAT1 labeling was accomplished by sulfhydryl modification using either [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) or N-ethylmaleimide (NEM). NEM is a membrane-permeant reagent, whereas MTSET is a membrane-impermeant reagent. In the presence of NaCl, transporter exposure to MTSET (1 mM for 5 min.) inhibited GAT1-mediated transport by 48%. Increasing the concentration of MTSET to 2.5 mM (5 min.) inhibited transport by 52%, and increasing the incubation time with MTSET to 20 min. (1 mM) inhibited transport by 66%. Loss of transporter function caused by MTSET modification was completely reversed by the reducing reagent dithiothreitol. MTSET treatment had no functional effect on the mutant GAT1 C74A, whereas NEM inhibited GABA transport mediated by GAT1 C74A by 15%. Thus, C74 is the only functionally significant cysteine residue that is accessible from the extracellular fluid. Sulfhydryl modification of the mutant GAT1 C74 is an efficient labeling method that paves the way for future experiments that seek to quantify GAT1 transporters expressed at the cell membrane.
Antifungal Effects of Combining Non-Drug Containing Liposomes with Amphotericin B Liposomes
Carla Noguera*, Davin Hahka, Jill Adler-Moore
Biological Sciences Department, Cal Poly Pomona
Introduction: Antifungal drugs, e.g. liposomal amphotericin B (AmBisome®), are used to kill fungi that cause life-threatening infections but the immune response of the body to the fungus is also needed to help resolve the infection. Aspergillus flavus and Aspergillus fumigatus infections often occur in immunocompromised patients and immunostimulating agents, such as non-drug containing liposomes (ndL), have been shown to help clear fungal infections in immunocompromised animals. It has not been determined if this effect of the ndLs is due to immunomodulation only, direct antifungal activity or if ndLs enhance the antifungal effects of AmBisome. We used microtiter broth assays to address the question of the antifungal activity of ndLs.
Methods: In vitro microtiter broth assays were used to determine the minimal amount of drug needed to inhibit (MIC) and kill (MFC) A. fumigatus and A. flavus. We tested AmBisome, ndLs and the combination of AmBisome and ndLs. We used 96 well microtiter plates with 1.7-3.8 x10^3 spores per well (A. flavus ) or 1.7-9.3x10^3 spores/well ( A. fumigatus )
Results: From several replicates (n= 15) of the microtiter broth assay, we determined that for A. flavus, the AmBisome MIC was from 0.78-3.1ug/ml. When AmBisome was combined with ndLs, the MIC was similar, ie. 1.56ug/ml, indicating that ndLs did not alter the antifungal activity of AmBisome for A. flavus. For A. fumigatus, the AmBisome MIC was from 0.58-2.34ug/ml. With the combination of AmBisome and ndLs, the MIC was also 0.58-2.34ug/ml. The AmBisome MFC for A. fumigatus was between 0.97-1.93ug/ml and for AmBisome plus, ndLs, it was slightly higher, i.e. 2.3-3.9ug/ml. Again, this showed that the ndLs had little to no effect on the antifungal activity of AmBisome for A. fumigatus. Importantly, the ndLs alone had no antifungal activity against either fungus.
Conclusions: The absence of any direct antifungal activity by the ndLs in the microtiter assay indicates that the beneficial effects of ndLs in the animals is probably due to their immunostimulating properties and that treating the animals with both AmBisome and ndLs does not interfere with AmBisome antifungal activity. For our future studies we will work with macrophages in vitro incubating them with fungal spores, AmBisome and ndLs to determine the antifungal and immunomodulating effects of these agents on yeast infections of macrophages. Research supported by NIH MBRS RISE (2 R25 GM061190-05A2).
Liposomes, Aspergillus, drugs
Pseudomonas aeruginosa and Staphylococcus epidermidis enhance Biofilm formation on Titanium Prosthetic Metals that Incorporate Boron
Alas, Steve — Pomona, Biological Sciences
Brelles-Marino, Graciela — Pomona, Biological Sciences
Marzano, Scott [U] — Pomona, Biological Sciences
Quijano, Jessamine [U] — Pomona, Biological Sciences
Prosthetics are the art and science of making artificial parts and implants for the human body. Human prosthetics are being utilized more frequently as the populations that require their use expand. These cohorts include an increasing elderly population, American soldiers returning with war injuries, and members of the general public who suffer serious injury from car collisions and sport related injuries. Human prosthetics commonly fail, however, due to chronic infection. Chronic infections that arise from biofilm forming bacteria are largely untreatable with antibiotics. Biofilm is a highly organized layer of microorganisms that firmly attach onto a surface using a extracellular polymer matrix (EPS). By testing new biometals comprised of titanium alloy with boron, we hoped to observe less biofilm propagation when compared to tranditional biometals currently used.
Using Pseudomonas aeruginosa and Staphylococcus epidermidis, two bacteria that commonly create biofilm on patient implants, we tested novel titanium alloys with regard to their susceptibility to biofilm propagation. The biometals examined were stainless steel (SS), commercially pure titanium (CPTi), Ti-6Al-4V (Ti64) and 3 novel titanium alloys that contain 0.05%, 0.4%, or 1.0% boron. Biofilm formation was analyzed using crystal violet staining and fluorescent microscopy. Ideal experimental conditions were obtained a biofilm reactor with TSB media, and incubation for 48 hours at 37˚C. Biofilm reactor experiments were performed with or without a constant infusion of growth media during biofilm formation.
The Ti64 metal alloy permits less biofilm formation than SS, CPTi and the titanium-boron alloys, by both Pseudomonas aeruginosa and Staphylococcus epidermidis, using both the flask method and biofilm reactor batch phase method. The same results were obtained with Pseudomonas aeruginosa in the method with constant flow rate in the biofilm reactor. These findings were corroborated using BacLight staining and fluorescent microscopy to verify the extent and viability of biofilm formed on the implant metals.
The Ti-6Al-4V alloy may be a better alternative to traditional metals, stainless steel and pure titanium, as a modern prosthetic biometal. Our findings indicate that the introduction of boron to the titaniu
Expression of Clostridial Autolysins and Their Role in Botulinum Neurotoxin Release
Dr. Wei-Jen Lin
Introduction: Clostridium botulinum is an anaerobic, Gram-postive bacteria that is responsible for the disease botulism, a deadly neurological and paralyzing disease. Botulism is usually derived by eating contaminated food. The ability to form spores gives them the advantage to survive in extreme conditions of drought, heat, or lack of nutrients and regrow when conditions are suitable. Clostridium botulinum produces the most dangerous toxin known to humans so far. It is one of the top biological risks that exists, which lead the organism to be strictly monitored and regulated by the government. The objective of this study is to clone and express autolysins from C. botulinum for in vitro characterization and study of their roles in the release of botulinum neurotoxin.
Methods: The first autolysin we chose to clone and characterize is the lysozyme CBO3274 which cleaves cell wall peptidoglycan. Gene specific primers were designed and the gene was amplified by the polymerase chain reaction and then cloned to a cloning vector. After verification of the gene sequence, the DNA was sub-cloned to an expression vector to create a poly-histidine-tagged fusion protein for expression and purification from an E. coli strain.
Results: PCR product show successful amplication of the target gene sequence. After introducing amplified PCR product into a cloning vector, DNA sequencing showed a 100% match by BLAST Analysis. Transformation of E. coli with pET30 expression vector and target insert was confirmed by gel electrophoresis. SDS-PAGE and Western blot analyses confirmed the presence and size of clostridial lysozyme in transformed E. coli.
Conclusions: We have successfully introduced clostridial genes in E.coli cells and induced expression of the proteins. For future studies, we want to characterize these clostridial proteins and investigate their function by introducing them in E. coli or other Clostridium species and observe cell leakage during early growth phases.
Comparison of the Efficacy of a Liposomal M2 Epitope Vaccine Prepared by Different Methods of Production
Natasha Ward1, Hana Kim1, Jill Adler-Moore1, and Suming Chiang2
1Department of Biological Sciences, California State Polytechnic University, Pomona, CA
2Molecular Express, Inc., Rancho Dominguez, CA
Introduction: The target for the present commercial influenza vaccines are the viral proteins, hemagglutinin (HA) and neuraminindase (NA), which are prone to yearly mutations (antigenic drift) or reassortment (antigenic shift) requiring new vaccines to be made every year. By incorporating the highly conserved region of the viral M2 protein (1-23aa) within a liposome, we can elicit significant protection against influenza strains, which have different HA and NA proteins. Our goal is to move this liposomal M2 epitope vaccine (L-M2e) into clinical trials and therefore, we needed to determine the optimum liposomal production method.
Methods: Liposomes were prepared as follows: 1) microfluidization using ethanol to solubilize the lipids (emicro), 2) sonication of a lipid film prepared with ethanol (efilm) or 3) sonication of a lipid film prepared with chloroform/methanol (c/mfilm). Female BALB/c mice (n=12/gp) were vaccinated with the liposome preparations (15ugM2e/dose) or liposome control (no M2e) (L-Con) on d0 s.c. and on d56 intranasally (i.n.). On d59, spleens were collected (n=5/gp). The splenocytes were used in an ELISPOT assay to determine cytokine production (IFN-γ for Th1 response vs IL4 for Th2 response). d62 blood (n=5/gp) was collected for ELISA antibody isotype titers. Remaining mice (n=7/gp) were challenged i.n. with 10LD50 H1N1 (A/PR/8/34) on d63 and monitored for morbidity.
Results: The mice vaccinated with efilm or c/mfilm L-M2e had 100% survival and mice given emicro L-M2e, had 71% survival; all but one mouse in the L-Con group survived (14%) (p<0.005 vs L-Con). Efilm L-M2e vaccinated mice lost the least weight and recovered faster compared to the other L-M2e groups although all the L-M2e groups lost significantly less weight (p<0.0001 vs L-Con) and recovered faster than the L-Con gp. In comparison, IL4 production was greater in the emicro L-M2e vaccinated mice (p<0.05 vs other groups) indicating upregulation of the Th2 response. All L-M2e formulations stimulated similar amounts of anti-M2e IgG1 and IgG2a.
Conclusions: The data support use of ethanol microfluidization as a scale-up method for making L-M2e vaccine needed for clinical trials. In addition, this technique allows the substitution of ethanol for the toxic organic solvents, methanol and chloroform, used currently in liposome production. Research supported by NIH MBRS RISE (2 R25 GM061190-05A2) and Cal Poly ARI.
Vaccine, Influenza virus, Liposomes
Expression of Clostridial Autolysins and Their Role in Toxin Release
Juan Ruiz, Wei Jen Lin
Clostridium botulinum is an anaerobic, Gram-positive bacteria that is responsible for the disease botulism, a deadly neurological and paralyzing disease. Due to the severe consequence that the botulinum neurotoxin (BoNT) poses to human and animal health, it is a major concern to public health officials and national defense agencies. The BoNT toxin is produced during cell growth but the mechanism through which the toxin is released is not known, but can occur through lysis of the cell. Our previous studies show that autolysins, which hydrolyze cell walls, may have caused cell leakage during early growth phases and led to the release of the toxin. In this study, we investigated the role of these autolysins in relationship to the early release of the toxin. The genomic sequence analysis showed thirty-four autolysins in C. botulinum, of which one-third are expressed in early growth phase based on our microarray study. The first autolysin we chose to clone and characterize is the lysozyme CBO3274 which cleaves cell wall peptidoglycan. The microarray study showed that this gene was found to be up-regulated during mid-log growth phase when toxin release was first detected. The gene was amplified by PCR and then cloned to a cloning vector. After verification of the gene sequence, the DNA was sub-cloned to an expression vector for expression and purification from an E. coli strain. Purified clostridial lysozyme will be added back to Clostridium strains and examined for its role in the release of the toxin during early growth phases.
Sensor based on Single-mode–Multi-mode–Single-mode (SMS) Fiber Structure
Michael Medrano and Ertan Salik
We investigated the sensitivity of an optical fiber sensor. An optical fiber is a thin thread of glass, and there are many different mechanisms for optical fiber sensors. The sensor we investigated is fabricated by splicing a short piece of multimode fiber between two pieces of single- mode fiber using a fusion splicer. This Single-mode–Multi-mode–Single-mode (SMS) structure works much like a modal Mach- Zehnder interferometer; light that travels in the single mode fiber splits its power into two modes of the multimode fiber. As light transitions back into the single-mode fiber, interference occurs. As stress or temperature is varied in and around the sensor, the effective refractive indices of the modes and to a lesser degree- the length of the path each mode travels must change, resulting in a change in relative phase of the modes and ultimately the transmission of light through the sensor.
Two sensors were created- both with a multimode fiber length of 10 cm. We used spontaneous emission of a semiconductor optical amplifier as the light source. Transmission was monitored with an optical spectrum analyzer. We placed the sensors on two fiber holders mounted on translation stages in order to determine the sensitivity to stress. Temperature sensitivity was determined by placing each sensor in a water- bath. We independently monitored the temperature with platinum-resistor type temperature probe.
The sensor showed sensitivity to both temperature and stress. However, sensitivity depends on the measurement wavelength. At a critical wavelength for the SMS structures, we confirmed that sensitivity was the highest. We predict about 1 microstrain stress resolution, and about 0.01 Kelvin temperature resolution. Because of the sinusoidal nature of the sensor response, the range of stress values and temperature values is rather limited, which requires further work.
MgO Composite Paints: Protecting Humans Against Harmful Bacteria
Authors: Jeanaye Mason, David I Zuniga, Daniel E Murrieta, Cal Poly Pomona
Mentors: Winny Dong, Tanya Faltens, Chemicals & Materials Engineering, Cal Poly Pomona
Traditional magnesium oxide (MgO) is an ionic crystalline solid with high bactericidal effectiveness. Other forms of MgO, amorphous MgO xerogels and aerogels can be synthesized via the sol-gel process. These novel structures are highly porous, giving them a greater surface area and concentration of structural defects than crystalline MgO. Increasing these two factors is expected to increase the bactericidal effectiveness of MgO.
In this work, we investigated the synthesis and properties of paints containing MgO xerogels and aerogels, which are of interest for aerospace and household applications. We have found that MgO reacts with water-based paint, causing it to prematurely solidify. To avoid this solidification, we investigated two anti-drying agents including glycerin and methanol.
We found that addition of 45-53wt% of glycerin to a paint of composition (list the wt% of each component in our paint) is effective in preventing solidification of the paint.
X-ray diffraction (XRD) analysis of the MgO paint composite indicates that MgO reacts with water in the paint to yield magnesium hydroxide (Mg(OH)2). In previous work, finely ground MgO powder suspended in aqueous solution was shown to maintain bactericidal effectiveness. In these previous studies, possible structural changes resulting from MgO reacting with water were not investigated.
Future work will focus on evaluating the bactericidal effectiveness of different magnesium oxides and hydroxides, the kinetics of the MgO reaction with water, and the incorporation of MgO into acrylic or oil based paints.
The Effect of Resveratrol on the Differentiation of hMSCs
Lindsay Peltz, Jessica Gomez, Negar Atashpanjeh, Frances Alencastro, and Yuanxiang Zhao
Resveratrol is a natural phytoalexin found in plants. It is a potent antioxidant and inflammatory suppressant that has been shown to extend lifespan in C. elegans, Drosophila, fish, and mice. It has also been implicated to have anti-tumorigenic and anti-adipogenesis effects in animal or animal cell models. All of these attributes may potentially apply to humans as well. However, very limited research has been conducted using human cells. We have studied the effect of short- and long-term resveratrol exposure on human adipogenesis, osteogenesis, and adult stem cell self-renewal by using human mesenchymal stem cells (hMSCs) as our in vitro cell model. hMSCs are multipotent adult stem cells that normally reside in multiple tissues including bone marrow and adipose tissue. They can differentiate into many different mature cell types, including but not limited to adipocytes, osteoblasts, chrondrocytes, and neurons upon receiving appropriate external stimuli. Our study demonstrated that Resveratrol had dosage dependent effect on the development of hMSCs. At concentrations greater than 10uM Resveratrol was found to be toxic to hMSCs. At concentrations equal or higher than 5uM, it inhibited both osteogenic and adipogenic differentiation of hMSCs, whereas at concentrations equal or less than 1uM it significantly promoted osteogenic differentiation of hMSCs but slightly inhibited adipogenic differentiation of these cells. Our results suggest that Resveratrol could be a beneficial non-dietary supplement for treating human conditions including obesity and osteoporosis, however, cautions need to be taken on the recommended daily dosage of synthetic Resveratrol in order to achieve a balanced outcome.