Recent Submissions

  • Surveying Mosses for Fungicidal Activity

    Yan, Stephanie; Department of Biological Sciences (Augusta University, 2019-05)
    The emergence of resistance to current fungicides is of serious concern because of the widespread diseases caused by fungi. One way to combat this problem is to discover new compounds that have antifungal properties. Plants are extensively attacked by fungi and many have evolved defenses, including fungicides and other compounds, such as a waxy cuticle, that make attack difficult. The mosses (Bryophyta) lack a cuticle. This makes them a likely group to survey for fungicidal activity because they may have additional chemical defenses. In this study, we made aqueous and ethanolic extracts from crushed mosses and tested their effect on the growth of the yeast Saccharomyces cerevisiae. These mosses were collected across a broad geographical range (locally, from Arkansas, and from Alaska) to test the hypothesis that resistance to fungal attack may correlate with the general suitability for fungal growth of the environment. Results include the demonstration of fungicidal activity in some, but not most, of the mosses. There was no correlation with geographical origin. Both solvents seem able to extract compounds that will suppress yeast growth. In addition, re-tests of some samples suggest that fungicidal properties may be lost during drying. Several of the mosses show strong enough antifungal activity that further investigation seems warranted.
  • The Study of 5-HT1D and 5-HT1F Receptor Interactions with G Proteins via BRET Analysis

    Trang, Amy; Department of Biological Sciences (Augusta University, 2019-05)
  • SYNERGISTIC CYTOTOXIC EFFECTS OF THE COMBINATION OF ERLOTINIB AND EXO2 ON HEAD AND NECK CANCER

    Thakkar, Parth; Department of Biological Sciences (Augusta University, 2019-05)
    More than 90% of head and neck cancer is head and neck squamous cell carcinoma1 (HNSCC). Currently, the treatment involves modern surgery, conventional chemotherapy, and radiation. However, targeting, the epidermal growth factor receptor (EGFR) has been shown to prove advantageous for patient survival. EGFR activation leads to cell cycle progression. Blocking the EGFR by an antibody results in the inhibition of the receptor, therefore inhibition of cell proliferation. This makes EGFR a prime target for anticancer therapy, specifically with tyrosine kinase inhibitors being looked at as a possible form of inhibition. The goal of this project was to hopefully use small molecule inhibitor EXO2 and an EGFR specific tyrosine kinase inhibitor, erlotinib, in a synergistic manner to fight against HNSCC. We hypothesize that the usage of both drugs together in a synergistic manner will prove to be a more effective treatment against HNSCC then using either drug alone. This study was done using cell cultures, MTT assay’s and western blot techniques, with cell cultures being done using the H6 cell line. The results from this study were found show a synergistic effect between EXO2 and erlotinib, supporting the hypothesis, but further studies into each drug individually as well s the interaction between both drugs must be done in future experiments.
  • Effect of night shift work on sleep memory

    Ravula, Ordain; Department of Biological Sciences (Augusta University, 2019-05)
    Sleep deprivation is a very common phenomenon world-wide and it has been known to affect motor and cognitive functions. In this multi-faceted study, we sought to assess the effect of sleep /deprivation on mood, cognitive performance, and memory, as well as any potential. Our sample was drawn from anesthesia residents at the Augusta University Medical Center. Our first focus was to determine the effects of sleep deprivation on mood, cognitive performance, and memory. Tasks were split amongst members of the research team, and I was charged with assessing memory retention and working memory via the Ospan test, a computerized test involving letter sequence memorization and mathematical problem solving. I administered the Ospan to residents at a baseline or control reading, then after their calls. If the residents had a night float (a stretch of calls over 5 days), then I would assess them after the first day of the float, right after the float, and 24 hours after the float. The higher the score on the Ospan, the better working memory and memory retention abilities were in that resident. Data collection began in April 2018 and continued into February 2019. For our secondary evaluation, we assessed how sleep deprivation affected gene expression, by looking at a set of genes known to be associated with sleep deprivation. These genes were: PER1, PER2, and ARNTL or BMAL. (Gene details explained in body paragraphs). Following similar protocol to the first study, expression was assessed before and after night calls and night floats. The testing involved sampling saliva from the residents who participated in the cognitive assessments in Part 1, isolating RNA from the samples, creating cDNA from the RNA, and then performing microarray analysis on the samples. To produce the cDNA samples from the RNA samples, reverse transcriptase PCR was used to create cDNA and amplify the number of copies of genes we had to work with. Microarray analysis was performed to measure the actual gene expressions of our genes of interest. These were certain genes which were previously found in another study by multiple researchers at the University of Surrey, in the UK (article title given below later) to affect sleep deprivation. These genes were then examined to determine if those specific genes increased, decreased, or maintained their expression, post-sleep deprivation.
  • Investigating the requirement of HOB1 on the sensitivity of Schizosaccharomyces pombe after exposure to various DNA damaging agents

    Qureshi, Arman; Department of Biological Sciences (Augusta University, 2019-05)
    DNA encodes the genetic information for the growth and development of living organisms. As such, it is inherently important that when damaged, the DNA is repaired efficiently and accurately. BIN1 encodes a protein that plays a role in genomic stability, specifically in cell cycle regulation, chromatin remodeling, and DNA repair. Previous research has shown that the protein Bin1 exhibits an inhibitory role in the double strand break repair pathway of non-homologous end joining (NHEJ). The homolog of BIN1, HOB1, is found in the fission yeast, Schizosaccharomyces pombe. To understand the role HOB1 has on yeast survival after damage, two strains of S. pombe, a wild type strain (WT) and a strain without HOB1 (hob1Δ), were exposed to various DNA damaging agents. Each treatment introduced a different type of DNA damage that would require different DNA repair pathways. The treatments included UV radiation, hydrogen peroxide, bleomycin, and cisplatin. After treatment with each respective agent, the % of surviving cells at multiple doses was analyzed and graphed logarithmically. The data collected supports the idea that the presence of HOB1 has a role on the survival of yeast after DNA damage. The WT strains survived better than the hob1Δ counterparts when exposed to hydrogen peroxide or bleomycin. When exposed to UV radiation or cisplatin damage, no significant difference is observed between the strains regarding survival.
  • The Mechanism of Inverse Agonists Binding to G-Protein Coupled Receptors, Histamine Receptor H1 and Histamine Receptor H2

    Patel, Shrey; Department of Biological Sciences (Augusta University, 2019-05)
    The thesis discusses the mechanism of an inverse agonist binding to receptors and how it is different from an agonist binding to the same receptor. The specific receptors that were focused on were histamine receptor H1 (HRH1) and histamine receptor H2 (HRH2) which are types of G-protein coupled receptors (GPCR). It is understood how an agonist binds to a GPCR and activates a signaling pathway within the cell, and that an inverse agonist can bind to the same receptor but elicit an opposite response. The current idea behind the mechanism of an inverse agonist is that it binds to the receptor, and the G-protein is not being recruited to continue the signaling pathway within the cell. The hypothesis was that the G-protein is recruited when the inverse agonist binds to the GPCR, but the G-protein would be in its GDP state or its inactivated state. To test the hypothesis, a luciferase assay was done in different conditions where the bioluminescence absorbance was measured and recorded to see if there was protein-protein activity between the GPCR and the G-protein. From doing multiple trials, it is still believed that the G-protein is not being recruited to elicit the signaling pathway when an inverse agonist binds to the receptors.
  • Solving a Problem at its Root: Is Aquatic Vegetation the Answer for Safe Composting in an Urban Brownfield?

    Mondeddu, Sheena R.; Department of Biological Sciences (Augusta University, 2019-05)
  • Investigating the Interaction between G Proteins and the 5-HT1E and 5-HT2C Serotonin Receptors Using BRET

    Little, Lauren; Department of Biological Sciences (Augusta University, 2019-05)
    G protein-coupled receptors (GPCRs) are important mediators in cellular signaling and are common targets of drug action. GPCRs are responsible for the transduction of extracellular signals into intracellular signals, mediated by G proteins of four subtypes: Gs, Gi, Gq, and G12/13. A thorough understanding of a signaling pathway involves determining which G protein is coupled to a signal-activated GPCR. In this project, a technique called Bioluminescence Resonance Energy Transfer (BRET) was used to measure the interaction between an activated GPCR from the serotonin (or hydroxytryptamine, 5-HT) receptor family, and G proteins from each subtype. The cDNA for serotonin receptors 5-HT1E and 5-HT2C was fused with the gene for a luminescent protein called Nanoluciferase (Nluc). Then, the receptor-Nluc DNA along with DNA containing a G protein tagged with a fluorescent protein (Venus) was transfected into mammalian cells for expression. Data from BRET assays suggest that the 5-HT1E receptor couples to the Gi/o subclass of G proteins upon serotonin activation, while the 5-HT2C receptor couples to the Gq subclass of G proteins. Profiling serotonin receptors will deepen our understanding of serotonin receptors, associated diseases, and the drugs that target them.
  • The Role of the JNK/Jun Signaling Node in ERα+ Breast Cancer Cell Survival and Autophagy

    Joseph, Carol; Department of Biological Sciences (Augusta University, 2019-05)
    A common treatment for estrogen receptor positive breast cancers is the use of selective estrogen receptor modulators such as Tamoxifen [1]. Unfortunately, 30-40% of patients experience relapse due to the development of antiestrogen resistance [2]. Autophagy, a process that is typically seen in cells that are exposed to a variety of stresses, is critical to the development of antiestrogen resistance and may play a key role in metastatic progression [3, 4]. To further combat antiestrogen resistance, a potential target for breast cancers is JNK (c-Jun N-terminal kinase), a member of the mitogen-activated protein kinase (MAPK) family. Our hypothesis is that JNK is a key regulator of autophagy and the emergence of autophagy-dependent antiestrogen resistant breast cancer. JNK-IN-8 was used for all experiments in this thesis proposal which tests the hypothesis that JNK1/2 is a key regulator of autophagy and the emergence of autophagy-dependent antiestrogen resistant breast cancer. The aims are to utilize ER+ breast cancer cells and determine (1) the effect of JNK1/2 inhibition on cell death under conditions of estrogen (E2) supplementation and antiestrogen treatment (+/- 4-OHT) and (2) the effect of JNK1/2 inhibition on autophagy. These studies have the potential to identify the JNK1/2/Jun signaling node as a molecular target for the improved treatment of breast cancer.
  • Effects of Chronic Alcohol and Glucose Exposure on Viability of Alveolar Macrophages

    Keller, Elizabeth; Department of Biological Sciences (Augusta University, 2019-05)
    The general population widely acknowledges the potential adverse health risks associated with the lifestyle factors of chronic alcohol abuse and obesity. These factors are manifested in the form of routine elevated blood ethanol and glucose levels, respectively. There is evidence which shows that the lungs are secondary organs affected by such physiological conditions. Healthy lungs are protected against infection and harmful airborne particles by macrophages—one of the working entities of the immune system. However, when these immune-responsive cells are compromised and unable to adequately perform normal functions, lung health may deteriorate. Therefore, a healthy pulmonary alveolar macrophage population is vital for the preservation of adequate lung function and for the prevention of respiratory infections and related complications. Chronic alcohol exposure and elevated glucose concentrations have been shown to suppress alveolar macrophage function, in turn possibly lowering the lungs’ first line of defense against foreign particles and infection. The objective of this study is to determine the effects of exogenous ethanol and increased glucose concentration on macrophage size and viability in relation to their compromised functionality within the human system through an in vitro study. This study tested alveolar macrophage response to conditions of elevated glucose levels, exogenous ethanol, and a joint treatment in a laboratory setting. Elevated glucose levels were representative of hyperglycemic conditions which often occur in states of obesity, while ethanol treatment simulated chronic alcohol exposure. NR8383 rat alveolar macrophages were grown in vitro in 25 cm3 flasks with each treatment. In addition to treatments, a control was maintained to ensure integrity of the experiment. Each conditional treatment was subjected to weekly viability testing and imaging for size analysis. The data was compared among the different treatment groups to gain a better understanding of how alveolar macrophages were affected by the specified conditions. The hypothesized theory anticipates an increased cell count in the glucose treated cells, but a decreased cell count in the ethanol and combination treatments. All three treatments were expected to yield smaller cell size, thus impairing macrophage function. Future studies plan to expose each treatment group to the bacterial agent lipopolysaccharide, or LPS, to test the initiated response of the macrophages and determine levels of functionality. LPS is commonly found on the outer membrane of gram-negative bacteria and is a reliable stimulant of the mammalian immune system. Differential responses of the treatment groups to LPS will indicate the overall health and functionality of the macrophages, helping to determine the effects that chronic alcohol and glucose exposure manifest on alveolar macrophages. Results from the current experiment will be analyzed to hypothesize clinically relevant responses.
  • Ecotoxicology of Yellow-Bellied Sliders (Trachemys scripta scripta) in Natural Wetlands

    Hammesfahr, Rachel; Department of Biological Sciences (Augusta University, 2019-05)
    Glyphosate is one of the active ingredients in many different herbicidal products such as Roundup. Preliminary research has suggested that glyphosate is a possible endocrine disruptor, can cause developmental defects, and is a potential carcinogen (Dallegrave et al., 2007; Daruich et al., 2001). Due to its potential harmful effects on different organisms, the researchers sought to monitor the levels of glyphosate in wetland areas. This was done by analyzing samples from a common indicator species, the yellow-bellied slider turtle, Trachemys scripta scripta. Research was done on turtles caught at Reed Creek Nature Center and Brick Pond Park. Physical measurements were taken, and blood was drawn from each turtle. Analysis of the glyphosate levels in the blood samples was completed using a glyphosate specific ELISA kit. Analysis from the kit showed detectable glyphosate levels in 34 out of 42 turtles tested, with concentrations ranging from 0.00 ppb to 0.59 ppb. No correlations could be found between glyphosate concentrations and the sex, mass, or age of the turtle, or the month the sample was collected, but glyphosate was found to be ubiquitous in turtles from both sampling areas. Further research could focus on different tissues such as fat or the scutes to see if glyphosate is stored in higher concentrations there than in the plasma. Other species, such as amphibians, living in the same areas could also be studied to determine if certain species are more susceptible to storing glyphosate than others.
  • Profiling G Proteins Using Bioluminescence Resonance Energy Transfer

    Farooq, Maheen; Department of Biological Sciences (Augusta University, 2019-05)
    GPCRs are receptors that act in signal transduction pathways via guanosine nucleotide-binding proteins (G proteins). Extracellular ligands act on GPCRs resulting in activation of one or more G protein subtypes (Gs, Gi/o, Gq/11 and G12/13) affecting the concentration of intracellular second messenger molecules ultimately altering cellular function. Cellular responses to external signals are typically studied indirectly by measuring concentration changes in second messengers. However, this approach can be problematic as many GPCRs can activate multiple G protein subtypes, and many second messenger pathways engage in “crosstalk”. To address this issue, we used Bioluminescence Resonance Energy Transfer (BRET) to directly measure coupling between 5-hydroxytryptamine (5-HT; serotonin) receptors and different G protein family subtypes. We co-transfected cells with plasmid DNA encoding the 5-HT2B or 5-HT4 receptors fused to the bioluminescent protein nanoluciferase (NLuc) as well as plasmid DNA containing G protein subtypes fused to the fluorescent protein Venus. In BRET assays, we found that mGsq couples to 5-HT2B and mGs couples with 5-HT4 in response to 5-HT activation. These results are consistent with the literature. Interestingly, initial studies suggest that activated 5-HT4 shows secondary coupling to mGsi highlighting the potential novel signaling pathways that can be elucidated using this technique.
  • SEQUENCE ANALYSIS OF ALU REPEATED ELEMENTS FOR PRIMATE PHYLOGENETIC TREE CONSTRUCTION

    Sood, Nitish; Mehra, Mehul; Department of Biological Sciences; University of California Berkeley; Bates, Christopher; Mittal, Anav; Augusta University (2019-02-13)
    Phylogenetic tree construction can be a particularly challenging and time-intensive process. This study employs a novel computational approach to phylogenetic tree construction, using the Alu repeating element, a SINE. Repetitive elements including Short and Long Interspersed Nuclear Elements (SINEs/LINEs) have successfully been applied as accurate tools for phylogenetic analysis, as they are predominately unidirectional and homoplasy-free. However, previous analysis of phylogenetic relationships using these repeating elements has been limited to a small number of isolated repeats among relatively few organisms. As a highly repetitive sequence, the Alu element and its associated subfamilies can provide detailed analysis on evolutionary divergence among species in the Order Primates. This study identified shared sequences as Alu repeating elements that were conserved in both location and base-pair sequence between the primate genomes of interest. These shared sequences, derived from the Genome Library at the University of California San Diego, were analyzed to construct individual phylogenetic trees for each of the 49 Alu subfamilies. As this method solely requires the sequence analysis of available primate genomes, this serves as a cheaper and more time-efficient approach to phylogenetic tree construction for the Order Primates relative to biochemical and anatomical analysis.
  • ADIPOSE HDAC9 DELETION PROTECT AGAINST DIET INDUCED OBESITY IN MICE THROUGH REGULATING ENERGY EXPENDITURE

    Hassan, Nazeera; Zarzour, Abdalrahman; Department of Biological Sciences; Department of Medicine; College of Allied Health Sciences; Kim, Ha Won; Weintraub, Neal; Augusta University (2019-02-13)
    Our group has previously identified histone deacetylase 9 (HDAC9) as a regulator of adipocyte differentiation, and its expression levels were elevated in diet induced obese (DIO) mice.� We also reported that global HDAC9 deletion protected mice against DIO through promoting beige adipogenesis. Here, we hypothesized that adipose HDAC9 correlate with human obesity similar to murine models, and its deletion is sufficient to protect against DIO. To test this hypothesis we crossed HDAC9 floxed mice with adiponectin-cre mice to generate adipose-specific HDAC9 knockout mice (AdipCre-HDAC9), which exhibited 30% less weight gain when fed high fat diet compared to control despite increased food intake, in association with increased energy combustion & O2 consumption, improved insulin sensitivity and glucose tolerance. However, unlike global HDAC9 deletion, this was not associated with increased beige adipogenesis nor increase in brown adipose tissue function. Interestingly, AdipoCre-HDAC9 mice fed normal chow diet didn�t exhibit altered energy expenditure nor weight differences when compared to littermate controls. These finding suggest that adipose HDAC9 regulate energy expenditure in response to high fat diet and can be a promising therapeutic target to combat obesity.
  • SYNERGISTIC EFFECTS OF THE COMBINATION OF ERLOTINIB & EXO2 ON HEAD AND NECK CANCER

    Thakkar, Parth; Department of Biological Sciences; Department of Oral Biology; Teng, Yong; Augusta University (2019-02-13)
    More than 90% of head and neck cancer is head and neck squamous cell carcinoma1 (HNSCC). Currently, the treatment involves modern surgery, conventional chemotherapy, and radiation. However, targeting, the epidermal growth factor receptor (EGFR) has been shown to prove advantageous for patient survival. EGFR activation leads to cell cycle progression. Blocking the EGFR by an antibody results in the inhibition of the receptor, therefore inhibition of cell proliferation. This makes EGFR a prime target for anticancer therapy, specifically with tyrosine kinase inhibitors being looked at as a possible form of inhibition. The goal of this project was to hopefully use small molecule inhibitor EXO2 and an EGFR specific tyrosine kinase inhibitor, Erlotinib, in a synergistic manner to fight against HNSCC. This study was done using cell cultures, MTT assay�s and western blot techniques, with cell cultures being done using the H6 cell line. The results from this study were found to be a preliminary success and will pave the way for future experiments in this area.
  • AN EXAMINATION OF MORAL PANICS: HOW THE FEAR OF SATANISM AFFECTED TABLETOP ROLE PLAYING

    Williams, Travis; Department of Biological Sciences; Department of Communication; Department of Anthropology & Philosophy; Johnson, Edgar; McClelland-Nugent, Ruth; Augusta University (2019-02-13)
    Moral panics around youth entertainment have been an occurrence as long as culture has been established. As long as youth entertainment has values that can be seen as going against the established values of the preceding generation, a moral panic could take place. The purpose of this research was to analyze how moral panics centered on youth entertainment begin and gain traction. To do this, the research was focused on the 1980s moral panic around tabletop roleplaying games, specifically�Dungeons & Dragons. By tracing the origin of the moral panic to the fear of cults and occult from the 1970s, we can find more context as to why some individuals believed that role playing games could cause adolescents to use the games as a style of dangerous escapism or as a gateway to the occult. To further understand this moral panic, an analysis of some of the major detractors of role playing games was done, as well as researching the role the media played in cultivating the moral panic. With a greater understanding on how moral panics begin and gain traction, this research can be used to compare and contrast other moral panics around youth entertainment.
  • EXAMINING THE EFFECTS OF AETOKTHONOS HYDRILLICOLA EXTRACT ON OXIDATIVE STRESS IN C6 CELLS

    Ward, Kayla; Department of Biological Sciences; Wiley, Faith; Augusta University (2019-02-13)
    Aetokthonos hydrillicola, a species of cyanobacteria, has colonized an invasive species of hydrilla in the lakes of the Southeastern United States. This cyanobacterium is suspected to cause Avian Vacuolar Myclinopathy (AVM). AVM is a neurological disease that affect birds. Bald eagles and American coots have been primarily studied and known to be affected by AVM. Symptoms of AVM consist of brain lesions, loss of basic motor skills, and the disease often leads to death. Extracts of A. hydrillicola are toxic to C6 cells, and this cell line is used as a model to examine the mechanism of toxicity. The aim of this research project is to understand the role of oxidative stress in A. hydrillicola cytotoxicity and determine if antioxidant compounds may protect the cells. Common oxidative stress inhibitors, Gingko biloba extract and selenium, have been tested in different concentrations in order to determine if oxidative stress is present and preventable. These compounds did not prevent toxicity in the C6 cells exposed to the cyanobacterial extracts. The presence of oxidative is currently being further investigated using a 2-7 dichlorofluorescin diacetate (DCFH-DA) assay, which indicates the presence of reactive oxygen species.
  • DEVELOPMENT OF TRANSGENIC ZEBRAFISH MODEL FOR INVESTIGATION OF THE FUNCTION OF MICROGLIA

    Sura, Survasha; Department of Biological Sciences; Department of Biochemical and Molecular Biology; Georgia Cancer Center; Rajpurohit, Surendra K; Augusta University (2019-02-13)
    Zebrafish have emerged as a powerful model organism for elucidating the development and function of microglia. Generation of new transgenic reporter lines and imaging tools strengthen the zebrafish model in microglia study�in-vivo. The aim is to develop a novel compound transgenic line to study the inflammatory process mediated by NF-kB in microglia cells. This novel compound transgenic line will establish a new model for microglia study. To generate the novel compound zebrafish transgenic model for microglia, we are crossbreeding microglia transgenic line zebrafish (Tg(mpeg1:mCherry) with the NF-kB Tg(6xNFkB:EGFP) transgenic progeny. We first generate a heterozygous F1 progeny which will be bred to generate an F2 homozygous progeny. Once the F1 progeny of the Microglia-NfkB transgenic line is developed, they will be crossbred to develop the Homozygous compound transgenic line. Fluorescent Microscopy will be used to screen the larvae generated from the breeding events. By developing the compound transgenic line, we are optimizing microglia isolation and sorting methodology by using the related antibodies as the marker. The NF-kB microglia transgenic line will provide a unique platform for drug screening to address microglial based ailments, thus furthering the understanding and treatment of human disease.
  • VACCINE PROLIFERATION IN THE FACE OF PUBLIC SCRUTINY

    Sripathi, Nishita; Department of Biological Sciences; Department of History, Anthropology & Philosophy; Turner, Wendy; Augusta University (2019-02-13)
    Each newly conceptualized vaccine has faced the same arguments over the last two centuries. A detailed examination of these several vaccines and their influences on the public will hopefully provide a better understanding of why the same arguments against vaccines continuously come up, even though each vaccine becomes widely used and celebrated. I supported my analysis by examining modern vaccine case studies and how those results may or may not skew the public reaction. By focusing on these two areas of research, I tried to understand the reasons behind persisted vaccine apprehension, even though there have been multiple and well-supported conclusions that vaccines are essential to a healthy human population. Perhaps by understanding the public�s fear, I can one day suggest alternate methods of vaccine �roll out� and introduction to the public.
  • PROFILING G PROTEINS USING BIOLUMINESCENCE RESONANCE ENERGY TRANSFER

    Farooq, Maheen; Department of Chemistry and Physics; Department of Pharmacology & Toxicology; Spencer, Angela; Lambert, Nevin; Okashah, Najeah; Augusta University (2019-02-13)
    GPCRs are receptors that act in signal transduction pathways via guanosine nucleotide-binding proteins (G proteins). Extracellular ligands act on GPCRs resulting in activation of one or more G protein subtypes (Gs, Gi/o, Gq/11 and G12/13) affecting the concentration of intracellular second messenger molecules ultimately altering cellular function. Cellular responses to external signals are typically studied indirectly by measuring concentration changes in second messengers. However, this approach can be problematic as many GPCRs can activate multiple G protein subtypes, and many second messenger pathways engage in crosstalk. To address this issue, we used Bioluminescence Resonance Energy Transfer (BRET) to directly measure coupling between 5-hydroxytryptamine (5-HT; serotonin) receptors and different G protein family subtypes. We co-transfected cells with plasmid DNA encoding the 5-HT2B or 5-HT4 receptors fused to the bioluminescent protein nanoluciferase (NLuc) as well as plasmid DNA containing G protein subtypes fused to the fluorescent protein Venus. In BRET assays, we found that mGsq couples to 5-HT2B and mGscouples with 5-HT4 in response to 5-HT activation. These results are consistent with the literature. Interestingly, initial studies suggest that activated 5-HT4 shows secondary coupling to mGsi highlighting the potential novel signaling pathways that can be elucidated using this technique.

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