Tag Archives: Life Sciences

The Effects of Nanomaterials on E. coli Growth

Gemma Clark, University of Utah

Nanomaterials are becoming increasingly common in the environment as they are engineered for technical purposes. Because nanotechnology is a relatively new technology, studies of nanomaterial toxicity effects are ongoing. For this study, Escherichia coli (E. coli) was used to determine the toxicity of coated zinc oxide, uncoated zinc oxide, iron oxide, and copper nanomaterials. Control conditions included no nanomaterials and zinc macromaterial. ATP fluorescence was used to monitor the growth curve of E. coli with and without nanomaterials, and culture methods were used to examine E. coli growth with varying concentrations of nanomaterials. The results so far show that as the concentration of nanomaterials increased, E. coli growth or ATP levels decreased and then increased again. These results were compared to E. coli growth without nanomaterials and E. coli growth with zinc macromaterial. Some nanomaterials may have had a greater impact on E. coli growth than other nanomaterials. These ATP fluorescence tests and culture methods will lead to future chemotaxis studies on the effects of nanomaterials as an attractant, repellent, or inert ingredient on E. coli movement.

Determining Kinetic Data for the APEH and ACY Pathway using GC-MS

David Coffman, Carson Cole, Weber State University

Proteases are a class of enzymes whose function is to cleave peptides into smaller amino acid chains or free amino acids. Two such proteases, Acyl Peptide Enzyme Hydrolase (APEH) and acylase (ACY), function in unison to cleave N-acylated amino acids from the N-terminal end of peptides so that these peptides can become further metabolized in the cell. In this pathway, APEH will first cleave an N-acylated amino acid from the end of a peptide. Then, ACY will remove the acetyl group from the amino acid so it can be recycled or further metabolized. While the enzymatic activity of APEH and ACY have been studied extensively alone, little has been done to study flux through the pathway. Understanding enzyme flux is useful since enzymes rarely work independently of one another in vivo but rather work in a coordinated fashion to perform their respective functions. This research illustrates a novel method to observe how both enzymes work together using Gas Chromatography Mass Spectrometry (GCMS). Using a selected N-acetylated dipeptide, the release of 2 free amino acids-Ala and Met- is detected by GC-MS and correlates to APEH and ACY activity, respectively. Using standard curves and time courses, the rate through APEH and ACY was determined. This method serves as a relatively simple way to study enzymes in vitro in an attempt to better understand the pathways function in vivo. In addition to measuring the flux of more than one enzyme in metabolic pathway, the use of this GCMS method has revealed that the enzymatic activity of the proteases in question do not function universally across different cell lysates. For example, it was determined that the rate of flux through APEH/ACY was 5 times faster in A549 cancer cell lysates than in normal blood lysates. The GCMS method described herein allows comparative studies of APEH/ACY flux and has potential to deepen the knowledge of how other these enzymes work together in both normal and disease states.

The Effects of High Glucose on the Oxidant Status of the Red Blood Cell

Mariah Richins, Jefferson Last, Dixie State University

Reactive oxygen species (ROS) are highly regulated in the body by antioxidants. When the regulation of ROS in the body is hindered, it can lead to oxidative stress and cell damage. The Glucose-6-Phosphate Dehydrogenase (G6PD) enzyme plays a part in reducing ROS and damage due to oxidative stress. It has been shown that the hyperglycemic conditions present in type 1 and type 2 diabetes mellitus impairs the activity of the G6PD enzyme, leading to oxidative stress and damage in endothelial cells1. Here, however, the activity of G6PD in red blood cells was specifically analyzed and determined to be lower in red blood cells treated with high glucose compared to controls. It is anticipated that upon addition of an anti-oxidant to the red blood cells treated with high glucose, the G6PD activity level will normalize. References 1. Zang, Z., Apse, K., Pang, J., & Stanton, R. C. (2000). High glucose inhibits glucose-6-phosphate dehydrogenase via cAMP in aortic endothelial cells. The Journal of Biological Chemistry, 275(51), 40042-40047. Doi: 10.1074/jbc.M007505200

Comparing Behaviors of Western Lowland Gorillas in Indoor and Outdoor Zoo Enclosures

Charlotte Brickwood-Figgins, University of Utah

Research has rarely been conducted past 2003 on comparing behaviors of zoo gorillas in different environments. The few relative studies suggest that, of course, naturalistic behavior is more common in naturalistic environments; ie. outside enclosures. “Exhibition of gorilla social groups in naturalistic habitats has been shown to…facilitate expression of species-typical behavior and enhance reproduction” (Lukas, K. [2003]). How does this species-typical behavior differ from an outdoor enclosure to an indoor enclosure? With the Hogle Zoo’s upcoming closure of the outside exhibit, during the winter months, I intend to draw a conclusion as to what differences the gorillas might display.

Homologous neurons play similar roles in reproductive-behavior circuits

Jess Breda, William Kristan, Kathleen French, Westminster College

A long-standing hypothesis in the field of neuroethology has been that organisms performing similar behaviors use similar underlying neuronal circuitry. Recently, though, studies focused on locomotion have shown that this may not necessarily be true. Work in sea slugs has demonstrated that homologous behaviors can be generated by a variety of neuronal connections. Our work looks at reproductive behaviors and asks whether those behaviors are mediated by the same neuronal connections in two species of leech. Research in Hirudo verbana has shown that reproduction is mediated by a neurohormone, hirudotocin, that is stored in and is released from a circuit of characterized neurons called Leydig Cells. A second leech, Macrobdella decora, has the same reproductive strategy as H. verbana, including a similar progression of behaviors. Using immunohistochemistry, we have identified Leydig Cell homologs in M. decora. Additionally, when we inject either M. decora or H. verbana with a hormone similar to hirudotocin, we observe reproductive-behavior output that is nearly identical. We intend to compare these species further by fully characterizing the Leydig Cell network using electrophysiology. These results will clarify the relationship between reproductive behavior and neuronal circuitry in leeches and give insight as to how neural circuits evolve across species.

Following the Nucleation Pathway of Gyroid

Maile Marriott, Laura Lupi, University of Utah

Nanostructures and the manufacturing of them are being researched for applications in areas such as controlled drug release, bio-sensors, solar cells, and data storage. The nanostructure known as the gyroid is particularly promising for application in these areas because of the continuous, fully connected channels that spiral through it periodically and uniformly. The gyroid can be manufactured through self-assembling block-copolymers or surfactants, but the mechanism for the assembly is not well understood. A better understanding of the parameters that control the formation of this structure will tell us how we can better control the formation of the gyroid structure. Our goal is to use molecular dynamics simulations to find the mechanism behind the formation of the gyroid in a model system. We were able to simulate its spontaneous formation and tested the efficiency of a number of order parameters, something that can distinguish between the gyroid and the surrounding mixture. As the gyroid forms, it passes through a transition state: the point where it has a 50% probability of forming or melting. The complexity of the gyroid structure makes it difficult to find an order parameter that can capture the structure at the transition state. However, by using committor analysis methods we were able to identify the transition state. We developed several order parameters that can distinguish gyroid from the isotropic mixture and, using Aimless Shooting and Maximum Likelihood Optimization, we ranked these parameters according to their effectiveness.

BACTERIOCIN PRODUCTION BY BIO-PROTECTIVE LAB CULTURES THAT INHIBIT LACTOBACILLUS WASATCHENSIS

Sophie Overbeck, Weber State University

Late gas defects in aging cheese result in significant losses to the manufacturer. Lactobacillus wasatchensis, a novel non-starter lactic acid bacteria (NSLAB), has recently been identified as an important cause of late gas defect. Controlling growth of this unwanted NSLAB may be done by incorporation of bio-protective LAB cultures (BP-LAB) into the cheese during manufacture, which could inhibit its growth during cheese aging. Previous research has shown that several BP-LAB cultures inhibit Lb. wasatchensis to varying degrees but the exact mode of inhibition has not been determined. Quantification of inhibition between BP-LAB cultures and Lb. wasatchensis was done using the spot test with the agar-flip method then measuring inhibition zones in comparison to time incubated. MRS agar with 1% ribose (MRS-R) was inoculated with each BP-LAB and incubated anaerobically at 35å¡C for 48 h to form a spot colony. Inoculated agar was flipped over and a Lb. wasatchensis strain swabbed on the exposed surface then incubated anaerobically at 25å¡C for up to 72 h. In addition, potential synergistic quantification of inhibition by co-BP-LAB strains was tested by taking 1 mL each of two different BP-LAB strains growing them together, and then following the previous described protocol. The five most inhibitory BP-LAB cultures were Lactobacillus rhamnosus LB3, Lactobacillus paracasei P-210, Lactobacillus brevis 13648, Lactobacillus casei F19, and Lactobacillus paracasei Lila. Four different co-cultures were tested LB3/ P-210, LB3/P-220, P-200/P-210, and P-200/P-220. No significant increases in the inhibition zones were observed when BP-LAB cultures were paired versus individual strains. Results confirmed that selected BP-LAB strains can inhibit growth of Lb. wasatchensis. Currently, we are trying to isolate the bacteriocins produced by these BP-LAB.

Effect of Organic Acids on Suppressing Growth of Lactobacillus wasatchensis

Ireland Green, Weber State University

Lactobacillus wasatchensis (WDC04) is a novel, slow growing, non-starter lactic acid bacterium (NSLAB) causing late gas formation in aging cheese, which results in significant economic losses to the producer. During cheese aging, organic acids can be produced by other NSLAB organisms or purposefully added to the cheese during manufacture. Organic acids are often used as preservatives. They are often found naturally in foods, generally don‰Ûªt affect flavor or product quality, and under acidic conditions, are able to enter bacterial cells effectively altering the cell‰Ûªs proton motive force. The effect of selected organic acids in their natural concentration range (2.5-560 mM) in Cheddar cheese was investigated for their ability to inhibit Lb. wasatchensis. Five organic acids (lactic, formic, propionic, citric and acetic) that can be produced by NSLAB organisms were selected and added at minimum, median, and maximum concentrations to individual wells of a 48 well plate containing MRS broth with 1% ribose (MRS + R) inoculated with WDC04. Growth rates were determined on a Tecan Infinite 200 PRO plate reader over 40 hours and results graphed on Excel. Both formic and citric acid showed inhibition of Lb. wasatchensis. As formic acid concentrations increased, the inhibitory effect also increased. The maximum concentration (100 mM) showed the most inhibition, the median concentration (63.15 mM) had an inhibitory effect between maximum and minimum, and the minimum concentration (26.3 mM) caused minimal inhibition. The addition of citric acid at the minimum (12 mM) and median (13.5 mM) concentrations showed similar inhibition. The use of these acids at concentrations normally found in Cheddar cheese is a potential antimicrobial measure to prevent or reduce late blowing in aging cheeses.

Examination of proteins bound to nascent DNA in mammalian cells using BrdU-ChIP-Slot-Western technique

Dominique Pablito, University of Utah

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA. Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples

Ferritin associations with Immune Cell Profile and Inflammatory Markers in Cross-Country Athletes

Bess Bauer, Weber State University

Objectives We evaluated the effects of different levels of stored iron (ferritin) within normal ranges on immune cell group production and inflammation markers in cross-country athletes. Research Methodology Forty-one NCAA division 1 cross-country athletes, ages 18 to 25 years old (Male: 19; Female; 22), had blood drawn at the beginning of the cross country season. Blood was collected from subjects and analyzed by complete blood count (CBC) at McKay Dee Hospital in Ogden, Utah. Enzymatic spectrophotometry was used to determine participants ferritin levels. Cytokines IL-1ë_ IL-6, IL10, GM-CSF, IL-5, and IL-4 were measured at baseline by the magnetic multiplex panel for Luminex TM platform (at University of Connecticut). Based on ferritin levels, participants were divided into two groups, high ferritin, and low ferritin. Student‰Ûªs t-test was used to compare cytokines and CBC mean difference between low and high ferritin groups. Pearson correlations were used to determine associations between cells groups and cytokines under low or high ferritin conditions. IBM SPSS statistics 22 software for Windows was used to analyze the collected data. Results Participants in the high ferritin group had higher levels of IL-1ë_ (p=0.04) and IL-5 (p=0.05) and eosinophils (p=0.02) when compared to the low ferritin counterparts. In contrast, IL-4 (p=0.04) was significantly higher in the low ferritin group. Moderate-strong correlations were found between eosinophils and cytokines; IL-1ë_ (r=0.64) and IL-5 (r=0.59) in the high ferritin group. Conversely, eosinophils from the low ferritin group correlated strongly with IL-4 (r=0.86) Conclusions In this study, it was observed that eosinophils correlated with different cytokines depending on the iron storage status. Our results are in accordance with previous studies showing that IL-1ë_ down regulates ferroportin (FPN) by increasing hepcidin levels which in turn decrease iron absorption. As iron storage in the form of ferritin increases, the need to absorb iron decreases inversely to IL-1ë_. Additionally, we observed increases in IL-4 in the low ferritin group. IL-4 has been observed to signal an increase in iron uptake (absorption) and mobilization. This is accomplished through transferrin (Tfn) expression. Furthermore, eosinophils and IL-5 were higher in the high ferritin group. IL-5 is a hematopoietic cytokine produced by eosinophils, it is possible that the increases in this biomarker were linked to the increases in red blood cell production observed on the high ferritin athlete group. Our results indicate that Iron storage status measured by ferritin levels may modulate cytokine release and immune cell profile.