Search

Inflammatory Bowel Disease (IBD), consists of both Crohn's colitis and Ulcerative colitis. IBD includes chronic inflammation and damage of the gastrointestinal tract. Crohn's colitis is specifically isolated to the large bowel and sometimes penetrates through all bowel layers. Ulcerative colitis, while also located in the large bowel, it is isolated to the epithelial layer of the intestines. Alpha Defensin 5 (HD5) is highly expressed in Crohn's colitis compared to Ulcerative colitis patients. HD5 is secreted by Paneth Cells to protect crypts from pathogenic bacteria by binding to the plasma membrane and lysing the cells. However, HD5 should not be found in the large intestine. Therefore, we hypothesize that HD5 is causing host cell death within the large intestine through a caspase-mediated pathway, contributing to symptoms of IBD. If HD5 is causing apoptosis through activating a caspase, then we should see a reduction in apoptosis when the cells are treated with HD5 and those caspases are blocked. If ZVAD, a broad caspase inhibitor, is added to cells prior to the addition of HD5, there should be a decrease in apoptosis. We measured the apoptosis using Annexin V and flow cytometry. If the HD5 is activating an apoptotic pathway, then by adding the ZVAD, there should be a decrease in apoptosis versus when there is no ZVAD added to the cells. This experiment will provide more of an understanding of how HD5 may be impacting disease progression within the colons of people with Crohn's colitis. [ABSTRACT FROM AUTHOR]

Inflammatory Bowel Disease (IBD) causes chronic inflammation of the gastrointestinal tract. IBD is diagnosed early in life of males and females and is considered a lifelong disease. The prevalence of IBD has significantly increased and is now considered one of the most common gastrointestinal diseases. In the US, approximately 1.6 million people have IBD, with 70,000 more people diagnosed each year. IBD that is present in the colon has two disease subtypes, Ulcerative Colitis (UC) and Crohn's Colitis (CC). Our lab has previously published that Human alpha-Defensin 5 (HD5) was found to be overexpressed in the large bowel of CC patients but showed normal expressions in the colon of UC patients. HD5 is an innate immune peptide that is commonly found in the small intestine. This peptide is encoded by the DEFA5 gene and is produced by crypt Paneth cells that line the small intestine. HD5 is antimicrobial and non-specifically kills bacteria, viruses, and fungi by compromising the microbial membrane. In the small intestine, HD5 keeps the environment sterile by opsonizing and destroying foreign pathogens. We are interested in determining the effects of overexpression of HD5 in the pathogenesis of CC. Our lab found that when Normal-Derived Colon Mucosa (NCM) cells were exposed to HD5 (unpublished data), there was increased cell death and decreased wound healing in cell culture. Therefore, we hypothesized that HD5 has at least one binding partner on colonic epithelial cells. In order to test our hypothesis, we have performed binding assays of HD5 to NCM membrane and cytosolic protein fractions and we will perform co-immunoprecipitations followed by amino acid sequencing to determine what HD5 is binding to on NCM cells. This information will allow us to better understand HD5's role in the pathogenesis of CC and how it contributes to the severity of the disease phenotype. [ABSTRACT FROM AUTHOR]

Mammalian histones are highly alkaline proteins found in the nuclei that organize DNA into chromosomes and regulate transcription. Cytochrome c is a heme protein that is found between the inner and outer mitochondrial membranes and functions to transfer electrons of the respiratory chain. We are investigating the interactions between histones and mitochondria and cytochrome C and mitochondria by using cell fractionation and Western Blotting to identify the histones and cytochrome C. The blocking step in the process coats the membrane with proteins or other molecules to reduce non-specific binding of the antibodies. Although 5% nonfat dry milk is widely used as a blocking buffer, we are testing different blocking buffers, as different blocking buffers yield different results, enhancing the signal or reducing it, depending on the specific antibodies used, and the optimal blocking buffer for a given antibody must be determined by experimentation. We use 5% nonfat dry milk (BLOTTO) as a control for all experiments, it is essential to use this variation of milk because it reduces background noise and helps produce good, clear bands. Since antibodies to histone H3 and cytochrome C are low-affinity and give a weak signal, we are therefore testing different blocking buffers, such as milk concentrations ranging from 1 to 5%, BSA ranging from 1 to 5%, and hemoglobin ranging from 1 to 5%, as well as gelatin at 3%, and PVP or PEG individually ranging from 1 to 4% and different combinations of the aforementioned blocking buffers to determine which blocking buffer gives the best detection signal to the histone and cytochrome C. Our preliminary results indicated that PVP/PEG combinations provided the strongest signal for both histone and cytochrome C, while gelatin tends to strip the proteins completely off of the blot and reduce the signal. [ABSTRACT FROM AUTHOR]

Seymeria is a relatively small genus of root hemiparasites in Orobanchaceae. Root hemiparasites retain the ability to photosynthesize while also establishing a xylem-to-xylem connection via the roots of host plants. The distribution area of Seymeria includes the eastern United States to Mexico. Pennell described 22 species and one subspecies in his 1922 revision of the genus and used this information to construct a rudimentary cladogram, primarily based on anther dehiscence. Turner confirmed 12 species, described three new species in 1982, and added another three new species 13 years later. His revision and new discoveries were focused on the species distributed throughout Mexico only. The most recent taxonomic work on the genus has been the addition of a new species in 2017. Phylogenetically, few Seymeria specimens have been included in studies focusing on the origins of parasitism and holoparasitism. This project seeks to use molecular techniques in addition to morphological characteristics to elucidate the evolutionary relationships within Seymeria. [ABSTRACT FROM AUTHOR]

In summer 2019, sediment samples were collected from five sites along the Mobile and Bon Secour Bays in the Gulf of Mexico. The characteristics of collected sediments (pH, temperature, salinity, conductivity, and TDS) were measured and PCR-based detection has been used to target fifteen genera of waterborne pathogenic bacteria in the samples. Currently, from about one third of the target pathogens tested, some pathogens have been detected in several samples. We expect our data to show the types and locations of pathogens in the Mobile and Bon Secour bay areas, which will help to predict the risk of waterborne pathogens under different salinity levels. [ABSTRACT FROM AUTHOR]

With a growing popularity of prescription and recreational use of marijuana, there has been increasing interest on the effects of this drug on early neurological development. Cannabidiol (CBD), the major non-psychoactive component of marijuana, has been approved by the FDA for use as a therapeutic agent for children two years and older2. Even so, there is limited research on its effect of CBD on early development. CBD oxidizes into Hu331, a chemical inhibitor of topoisomerase-II beta (TOP2B) and potential anti-cancer therapy. TOP2B relieves DNA supercoils during replication and transcription and is also required for proper neural development. Previous studies in our lab have shown that early Hu331 exposure results in reflex deficits and axon guidance defects. In 2019, our lab started to investigate the effect of CBD exposure on early development. Our data suggests that CBD exposure causes neuro-development defects similar to those induced by with HU331 exposure. Our findings have shown that axon guidance is impacted by both Hu331 and CBD exposure. This study further explores the impact of CBD and Hu331 on axon guidance, neural differentiation and cell death. [ABSTRACT FROM AUTHOR]

Zebrafish have a long-standing reputation in molecular biology for being an outstanding model to view vertebrate organisms' development. The zebrafish's structural development is relatively rapid compared to higher vertebrates, yet their development is just as complex. During the zebrafish gastrula period (5.25-10 h), during embryogenesis, cell movement and tissue expansion occur, creating the three primary germ layers: ectoderm, mesoderm, and endoderm. Within the endoderm and mesoderm, Collagen, type II, alpha 1a (col2a1a) is expressed in several structures such as the fin, floor plate, head, and skeletal system. A subset of mesodermal cells, known as the sclerotome, gives rise to the axial skeleton, consisting of cartilage, bone, and associated connective tissues. This study aims to understand how different cell fates in the developing axial skeleton are formed. Two genes expressed within the sclerotome, twist1b and twist2, were knocked down via morpholino injection at the single-cell stage to disrupt sclerotome development. Using in situ hybridization, we studied the expression of the col2a1a gene and found it is involved in forming the bone, tendon, and cartilaginous structures within the axial skeleton. Specifically, we found that twist1b and twist2 genes are required for proper col2a1a expression within these developing tissues. To follow-up on these findings, further investigation on the BMP pathway related to osteogenic differentiation will provide more data on the bone and tendon differentiation pathway. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is an opportunistic pathogen renowned for inducing cryptococcal meningoencephalitis in immunocompromised individuals. This basidiomycetous, obligate-aerobe yeast is also a robust model organism for studying fungal genetics. Despite prior research, therapeutics selective against C. neoformans are marked by limited options and risk of severe complications. Alternative drug designs are desirable, and molecular biological techniques are being used to spearhead research into novel drug targets. One such potential drug target is a mitochondrial protein -- DNA Polymerase Gamma (PolG). To probe the necessity of the cryptococcal PolG ortholog (CnMIP1), I have established a transient CRISPR-Cas9 system capable of inducing gene recombination via Homology Directed Repair (HDR) in a C. neoformans serotype D strain. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 is a gene editing technique that uses an RNA guide to induce precise DNA damage. Conventional gene editing and chromosomal integration in C. neoformans has relied upon biolistic techniques; CRISPR poses to be a more precise and cost effective method allowing for easier gene manipulation in vivo. Accordingly, a transient CRISPR system with guide RNAs targeting exon2 of cryptococcal URA5 was developed to test for recombination with a geneticin resistance (NEOR) gene in an easily screenable target gene. Trials have yielded preliminary evidence for site-specific recombination and gene disruption, resulting in dual NEOR and ura5Δ phenotypes. This system is being tailored to target the CnMIP1 promoter for recombination with a gene cassette containing a NEOR gene, a copper-regulatable CTR4 promoter, and a GFP expression marker. To accomplish this, a multi-fragment ligation technique was used to rapidly assemble the cassette in a small vector. This will be used as an HDR template for CRISPR-induced replacement of the CnMIP1 endogenous promoter, so that gene expression may be conditionally regulated such that CnMIP1 necessity can be genetically ascertained. [ABSTRACT FROM AUTHOR]