Our goal was to design a method to express soluble folded major histocompatibility complex (MHC) proteins using human cell line HeLa lysate with the novel 1-Step Human In Vitro Protein Expression by Thermo Scientific in the presence of β2 microglobulin (β2m) and antigenic peptide.
We confirmed that the soluble protein MHC-A2.1 could be successfully attached to the Luminex magnetic beads and detected using the primary antibody anti-GST and the detection antibody goat mAb mouse PE. The average net MFI of the attached pA2.1-bead complex was 8182. Biotinylated A2.1 MHC complexes pre-folded with β2m and FLU M1 peptide (A2.1 monomers) were also successfully attached to Luminex magnetic beads and detected with BB7.2. The average net MFI of the detected A2.1 monmer-bead complexes was 318. The protein MHC complexes were multimerized on magnetic beads to create MHC tetramers and detected with BB7.2, PE labeled monoclonal antibody, via median fluorescent intensity with the Luminex platform. Varying protein, β2 microglobulin (β2m), and peptide concentrations were tested in a number of MHC-A2.1 protein refolding trials. Different antigenic peptides and attachment methods were also tested. However, none of the MHC-A2.1 protein folding and capture trials were successful. Although MHC-A2.1 complexes and recombinant MHC molecules could be attached to Luminex magnetic beads and be detected by Luminex arrays, soluble protein A2.1 could not be successfully expressed, refolded, captured onto Luminex beads, and detected. All refolding trials resulted in a net MFI of <25. The failed refolding and capture trials of A2.1 lead to the conclusion that human cell line HeLa lysate cannot be used to properly fold MHC molecules. However, efforts to refold the complexes onto Luminex magnetic beads are ongoing. We are also using the baculovirus expression system to refold soluble A2.1 lysate onto peptide-bead complexes.

Bioprospecting is the exploration of biodiversity in search of new resources with potential economic value. Since the colonial era, bioprospecting has led to the innovation of many valuable medical and commercial products. However, the extraction of these natural resources has historically been linked to the exploitation of indigenous populations, known as biopiracy. This review of the Maya ICBG Biopiracy Controversy provides an in-depth exploration into the delineation between bioprospecting and biopiracy to reveal that the unfounded claims of biopiracy halted a potentially successful bioprospecting project.

Background: The American Heart Association has created an Official 2012 Hands-Only CPR Instructional Video that is approximately one minute in length and has been viewed over 600,000 times on YouTube. Objective: To evaluate the video's effectiveness in teaching adolescents aged 12-17 hands-only CPR. Methodology: The study took place in the Phoenix metropolitan area. Study participants were recruited from high schools, junior high schools and the Tempe Boys & Girls Club. The 100 study participants took a short, four question survey and watched video on either a laptop computer or video projector. Participants were then subjected to a cardiac arrest mock scenario in which they were tested on calling 911, compression rate, compression depth, and hand placement using a Lifeform CPArlene Recording Manikin. In analysis of the data, subjects were separated into four groups: 12-14 year olds (middle school aged) who had previous CPR training (MSG-T), 12-14 year olds with no previous training (MSG-U), 15-17 year olds (high school aged) who had previous training (HSG-T) and 15-17 year olds with no previous training (HSG-U). Results: Every study participant performed hands-only CPR during the mock scenario. Between the two middle school-aged groups, the MSG-U was more likely to call 911 during the mock scenario (P<0.05). There were no significant differences in compression rate and depth between the MSG-U and MSG-T. Between the two high school-aged groups, the HSG-T was more likely to call 911 during the mock scenario (P<0.05). There was no significant difference in compression rate between the HSG-T and HSG-U groups. The HSG-T compressed the chest significantly deeper than the HSG-U group (P<0.05). The HSG-T was the only group to statistically be on par with the AHA recommended 100 compressions/minute (P<0.05). All other groups were significantly below the 100 compressions/minute standard. No groups were statistically on par with the AHA recommended compression depth of two inches. Conclusion: The Official 2012 Hands-Only Instructional Video should not be used as a definitive training tool to teach school-aged adolescents hands-only CPR. This video, as well as other similar training videos, would be useful as introductory tools for children 12-14 years of age or as a refresher for older children who have received previous training.

This study focuses on the death and mourning practices that surround the passing of a pet. Through the compilation of previous research and localized polls of Arizona State University students, I attempt to better understand the impact that the loss of a pet has on its owners and those involved. More specifically, I analyze the role of a pet and how a pet's death can influence and shape aspects of our society in regards to euthanasia, grief and mourning. Once a full analysis of pet death in the United States is completed and polling results are discussed, the study will also briefly discuss the practices and attitudes found in Japan and the United Kingdom for cross-cultural comparison.

This long form creative nonfiction essay gives insider details on working in an emergency room as a medical scribe. The most pertinent topic is death and how the author copes with seeing patients die on a regular basis. Other topics are emergency room procedures, specific diagnoses and treatments, as well information on the other personnel in an emergency room.

In vitro measurements of cellular respiration have proven to be key biomarkers for the early onset of tumor formation in certain pathological mechanisms.1 The examination of isolated single cells has shown promise in predicting the onset of cancerous growth much earlier than current methods allow.2 Specifically, measurements of the oxygen consumption rates of precancerous cells have elucidated outliers which predict the early onset of esophageal cancer.2 Single cell profiling can fit in to current pathology studies and can serve as a step along the way, much like PCR or gel assays, in detecting biomarkers earlier than current clinical methods.3 Measurement of these single cell metabolic rates is currently limited to 25 cells per experiment. It is the aim of this project to increase throughput from 25 cells to 225 cells per experiment via the implementation of new hardware and software which fit with current methods to allow the same experimental structure. Successful implementation of such methods will allow for more rapid and efficient data collection, facilitating quantitative results and nine times the yield from the same experimental manpower and funding. This document focuses on the implementation ultra high density (UHD) hardware consisting of a pneumatic molar design, angular adjustment features and a mechanical Z-stage. These components have produced the most encouraging results thus far and are the key changes in transitioning to higher throughput experiments.

AMPylation is a post-translation modification that has an important role in the survival of many bacterial pathogens by affecting the host cell's molecular signaling. In the course of studying this intercellular manipulation, there has only been modest progression in the identification of the enzymes with AMPylation capabilities (AMPylators) and their respective targets. The reason for these minimal developments is the inability to analyze a large subset of these proteins. Therefore, to increase the efficiency of the identification and characterization of the proteins, Yu et al developed a high-throughput non-radioactive discovery platform using Human Nucleic Acid Programmable Protein Arrays (NAPPA) and a validation platform using bead-based assays. The large-scale unbiased screening of potential substrates for two bacterial AMPylators containing Fic domain, VopS and IbpAFic2, had been performed and dozens of novel substrates were identified and confirmed. With the efficiency of this method, the platform was extended to the identification of novel substrates for a Legionella virulence factor, SidM, containing a different adenylyl transferase domain. The screening was performed using NAPPA arrays comprising of 10,000 human proteins, the active AMPylator SidM, and its inactive D110/112A mutant as a negative control. Many potential substrates of SidM were found, including Rab GTPases and non-GTPase proteins. Several of which have been confirmed with the bead-based AMPylation assays.

Ultrasound is a sound wave that produces acoustic pressure and is most commonly known as a noninvasive technique for bodily imaging. However, high-intensity focused ultrasound can be used for noninvasive physiotherapy. An example of this the treatment of tumors in the kidneys, as the sound waves of HIFU interacts with tissues in the body. For this thesis, the necessary parameters for ultrasonic stimulation of the central nervous system in rats were characterized.

Anole lizards that inhabit the islands and mainland of the Caribbean basin have evolved morphological traits adapted to the microhabitat that they occupy. The anoles on these islands have been characterized as "ecomorphs" or morphologically and behaviorally-adapted groups, including: crown-giant, trunk-crown, trunk, grass-bush, twig, and trunk-ground. Ecomorphs display morphological features that are specifically adapted to the habitat that the anole occupies. One key morphological difference is tail length. While the anoles Anolis carolinensis and A. sagrei have similar ratios of tail length versus snout-to-vent length (SVL), they occupy different microhabitats. Specifically, A. carolinensis inhabits trunk-crown habitats while A. sagrei is found in trunk-ground regions. In this study, I focused on analysis of the caudal vertebrae of these two species, to determine if the structure of the osteological elements reflected differences in microhabitat adaptation. Skeletal preparations reveal that A. carolinensis have 40 \u2014 46 caudal vertebrae, and A. sagrei have 38 \u2014 49 caudal vertebrae. Transverse processes are present in Ca1-8 in A. carolinensis whereas transverse processes in A. sagrei span from Ca1-42 vertebrae. Ca6\u201440 have autotomy planes in A. sagrei, whereas only Ca8\u201417 have autotomy planes in A. carolinensis. These findings indicate that A. carolinensis are limited in the ability to autotomize their tail compared to A. sagrei. A. carolinensis, living higher in the trees than A. sagrei, might incur a greater impairment of locomotor function if autotomized. There appears to be no differences between males and females of both species in respect to vertebrae lengths. Differences between A. carolinensis and A. sagrei in terms of vertebral length are found in Ca12-15, 29-30, 34, and 37. The finding indicates that almost all caudal vertebrae between A. carolinensis and A. sagrei have similar relative lengths, but seven vertebrae have statistically significant differences. The biological significance of the findings is not clear, but functional and myological studies may help elucidate the reason of the observed differences.

Tumor associated microglia-and-macrophages (TAMS) may constitute up to 30% of the composition of glioblastoma. Through mechanisms not well understood, TAMS are thought to aid the progression and invasiveness of glioblastoma. In an effort to investigate properties of TAMS in the context of glioblastoma, I utilized data from a PDGF-driven rat model of glioma that highly resembles human glioblastoma. Data was collected from time-lapse microscopy of slice cultures that differentially labels glioma cells and also microglia cells within and outside the tumor microenvironment. Here I show that microglia localize in the tumor and move with greater speed and migration than microglia outside the tumor environment. Following previous studies that show microglia can be characterized by certain movement distributions based on environmental influences, in this study, the majority of microglia movement was characterized by a power law distribution with a characteristic power law exponent lower than outside the tumor region. This indicates that microglia travel at greater distances within the tumor region than outside of it.