The last decade for the Medical College of Georgia has yielded strategic, phenomenal growth in educational, research and clinical initiatives in diseases affecting every family in Georgia and the United States.

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Recent Submissions


    Crouch, John Jeffrey; Department of Advanced Studies and Innovation (Augusta University, 2019-05)
    Mentoring has been shown to have a positive impact on student outcomes such as attendance, behavior, and overall connectedness to school. Through strengthening relationships with a non-familial adult, mentoring has also been shown to have a positive impact on student interactions with other adults within the school environment. However, there are many logistical considerations that can adversely impact the implementation of a school-based mentoring program. This study began as a mixed methods study intended to examine the impact of a community-based mentoring program on student discipline referrals and absences. During the course of the study, the scope and methods shifted to become a qualitative study that focused on the implementation of an after-school mentoring program for middle school students. The authors employed a case-study methodology using a variety of data collection methods including interviews with mentors and administrators, a focus group with the mentees, and repeated observations of the mentoring sessions. Thematic content analysis revealed six themes: goals, experiences, perceptions, relationships, challenges to implementation, and sustainability and improvement. Findings suggest that the faculty and staff had a high level of confidence in their leadership which was likely to positively impact the mentoring program, as they were more likely to trust his decisions and work diligently to ensure that his goals for the program were met. Should a mentoring program be implemented, our findings indicated that time and prioritization are imperative to its success. Keywords: mentoring, relationships, leadership, school-based, improvement
  • Genetic Modeling and Pathophysiological Analysis of FAM109A, a Putative Human Disease Gene

    Ates, Kristin Marie; Department of Neuroscience and Regenerative Medicine (Augusta University, 2019-05)
    A critical barrier in the treatment of endocytic diseases is the lack of information and understanding of the in vivo mechanisms of endocytosis. Part of this is due to the diverse array of endocytic adaptor proteins that have not yet been studied. We address this by investigating a key endocytic adaptor protein, FAM109A, which interacts with OCRL1, a causative gene for Lowe syndrome. Previous in vitro studies have identified FAM109A as a regulator for endosomal trafficking, particularly in the recycling of receptors in endosomes and sorting of cargo to lysosomes, based on knock-down studies. Here we conduct the first study into the developmental and physiological functions of FAM109A in vivo, utilizing the zebrafish model. We find that depletion of both zebrafish orthologs, zFAM109A and zFAM109B, in our maternal-zygotic homozygous mutant models (AB mutant) disrupts fluid-phase endocytosis and ciliogenesis in the pronephros. Partial knockdown of OCRL1 in the AB mutants exacerbates the endocytosis deficit, confirming that OCRL1 and FAM109 proteins are linked in a common endocytic pathway. In addition, we discover that zFAM109A/B mutant animals exhibit reduced jaw size and delay in chondrocyte maturation, indicating a novel role for zFAM109A and zFAM109B in craniofacial development. This is consistent with the phenotype in a patient within the NIH’s Undiagnosed Diseases Program (UDP). The UDP patient carries a de novo arginine (R) to cysteine (C) mutation (R6C) in FAM109A and presents with craniofacial abnormalities, developmental delay, auditory and vision impairments, and renal dysfunction. Expressing zFAM109A with the R6C mutation in zebrafish exacerbated craniofacial deficits, suggesting that the R6C allele acts in a dominant-negative manner. Together, these results show that FAM109A is involved in fluid-phase endocytosis and ciliogenesis in vivo. Moreover, we provide further insight into the potential pathogenesis of a UDP patient’s disease in association with a de novo mutation in FAM109A.

    Ibrahim, Mohammed Mahmoud Labib; Biomedical Sciences (Augusta University, 2019-05)
    A causal link between chronic inflammation and tumorigenesis is now well established in the literature with a great deal of supporting evidences from genetic, epigenetic, pharmacological and epidemiological perspectives. In particular, inflammatory bowel diseases represent an important risk factor for colon cancer development. Moreover, it seems that even sporadic colon cancers that do not develop as a complication of chronic colitis are also driven by inflammation. However, the molecular mechanisms behind inflammation-mediated colon tumorigenesis have remained largely unknown. Colitis associated cancer development is thought to be multifaceted due to a combination of genetic and epigenetic aberrations. Recently, epigenetic alterations -particularly aberrant DNA methylation- have gained great attention in cancer biology and have been observed to play a key role in the pathogenesis of inflammation-associated tumors; especially in colitis-associated cancer. IRF8, a key transcription factor originally identified in myeloid cells, has been reported to play a crucial role in myeloid cells differentiation and immune response regulation. IRF8 deficiency is associated with deregulation of myeloid cell differentiation and accumulation of immature myeloid subsets phenotypically and functionally resemble MDSCs (Myeloid Derived Suppressor Cells). IRF8 is thought to function as a tumor suppressor and was found to be silenced in different types of cancers including colon cancer. Myeloid derived-IRF8 has been extensively studied. However, the role of epithelial-derived IRF8 in colon inflammation and colon cancer initiation remains a point to be addressed. In this study, we generated conditional Irf8cKO mice in which IRF8 is specifically deleted in colon epithelium. Irf8cKO mice exhibit a more aggressive pattern of colitis associated cancer with higher tumor incidence and severe loss of body weight. Additionally, we provide evidence that chronic inflammation promotes the accumulation and infiltration of CD11b+Gr1+ MDSCs, which plentifully secrete IL10 in colon tissue. IL10 then induces STAT3 phosphorylation and nuclear translocation to bind to Dnmt1 and Dnmt3b promoters to upregulate their expression, leading to DNA hyper-methylation at the Irf8 promoter to silence IRF8 expression in colonic epithelial cells and promote colon tumorigenesis. Collectively, our data pinpoint the MDSC-IL10-STAT3-DNMT3b-IRF8 axis as a novel bridge between chronic inflammation and colon cancer formation.

    Tucker, Donovan; Tucker, Lorelei; Department of Neuroscience and Regenerative Medicine (Augusta University, 2019-05)
    Neonatal hypoxic ischemic encephalopathy (HIE), initiated by hypoxic-ischemic (HI) injury to the brain in the perinatal period, is a leading cause of infant mortality and disability. HI damage to the developing brain triggers a complex pathology, initiating with mitochondrial insult, which culminates in neuronal cell death. Photobiomodulation (PBM), the application of near-infrared light, is an experimental neuroprotective strategy targeting the activity of mitochondrial cytochrome c oxidase (CCO), but its effect on HIE is unknown. This work was designed to shed light on the effect of PBM on a neonatal rat HI injury model. Postnatal day 10 mixed-sex pups underwent HI insult followed by 7 daily PBM treatment sessions via a continuous wave diode laser (808 nm). HI pups suffered significant ipsilateral hemispheric brain shrinkage and substantial cell death in the cortex and hippocampal CA1 and CA3 subregions. PBM treatment reduced neuronal cell death in the cortex and hippocampal subregions and reduced hemispheric brain shrinkage. HI pups displayed impaired motor function and spatial learning and memory which was ameliorated by PBM. Blood-brain barrier integrity was compromised in HI animals, as evidenced by reduced extravasation of Evans blue, but was reversed by PBM. PBM also mitigated microglial activation and upregulation of pro-inflammatory cytokines in HI pups. PBM treatment induced robust reduction in oxidative damage markers and protein carbonyl production in the cortex and hippocampus. Investigation of mitochondrial function revealed that PBM markedly attenuated mitochondrial dysfunction and preserved ATP production in neonatal HI rats. Furthermore, PBM treatment profoundly suppressed HI-induced mitochondrial fragmentation. PBM administration reduced activation of pro-apoptotic caspase 3/9 and TUNEL-positive neurons in HI pups. Finally, we demonstrated that the neuroprotective action of PBM could be reversed in a primary hippocampal neuronal OGD model by application of low-dose KCN, a CCO inhibitor. Taken together, our findings demonstrated that PBM treatment contributed to a robust neuroprotection via attenuation of mitochondrial dysfunction, oxidative stress, and neuronal apoptosis in the neonatal HI brain. Additionally, we demonstrated that these effects are, in part, mediated by modulation of CCO activity. This suggests that PBM may offer a promising role as a potential treatment strategy for HIE.
  • In search of genetic mutations for familial keratoconus

    Khaled, Mariam Lotfy; Department of Cellular Biology and Anatomy (Augusta University, 2019-05)
    Keratoconus (KC) is the most common corneal degenerative disorder and a leading cause of corneal transplantation in developed countries. KC is a multi-factorial disease with involvement of genetic, environmental, and hormonal factors. Although KC has been widely studied, the main cause of the disease and the molecular mechanism remain unknown. We aimed to study the molecular genetics of KC via utilizing next-generation sequencing technology including RNA-Seq, whole exome sequencing, and whole genome sequencing. We used RNA-Seq to study the KC-affected corneal transcriptome. We identified 436 coding RNAs and 584 lncRNAs with differential expression in the KC-affected corneas with a |fold change| ≥ 2 and a false discovery rate ≤ 0.05. Pathway analysis, using WebGestalt, indicated the enrichment of the genes involved in the extracellular matrix, protein binding, glycosaminoglycan binding, and cell migration. Co-expression analysis revealed 296 pairs of genes with significant KC-specific correlations. The RNA-Seq data analysis highlighted the potential roles of several genes (CTGF, SFRP1, AQP5, lnc-WNT4-2:1, and lnc-ALDH3A2-2:1) and pathways (TGF-β, WNT signaling, and PI3K/AKT pathways) in KC pathogenesis. Next, we used whole genome and exome sequencing to figure out the causal mutation(s) in a four-generation KC family with a linkage locus on Chr5q14.3-q21.1. We found a missense mutation in the phosphatase domain of PPIP5K2 (c.1255T>G, p.Ser419Ala). We found another missense mutation in the same domain of PPIP5K2 (c.2528A>G, p.Asn843Ser) in a second KC family. PPIP5K2 is a bifunctional enzyme involved in the inositol phosphate metabolic pathway. In vitro functional assays indicated the impact of the identified mutations on the enzymatic activity of PPIP5K2. PPIP5K2 expresses at a higher level than its homolog PPIP5K1 in both human and mouse corneas. A transgenic mouse model with the loss of phosphatase activity and elevated kinase activity of Ppip5k2 exhibited corneal structural abnormalities emphasizing the important role of PPIP5K2 in the homeostasis of corneal integrity. This study advances our knowledge of KC genetic etiology and helps in identifying a potential therapeutic target for KC.
  • The c-MYC oncogene deregulates global DNA methylation and hydroxymethylation to control genome-wide gene expression for tumor maintenance in leukemia/lymphoma

    Poole, Candace Jean; Biomedical Sciences (Augusta University, 2019-05)
    Aberrant DNA methylation is a characteristic feature of tumor cells. However, our knowledge of how DNA methylation patterns are established and maintained to contribute to tumorigenesis is limited. Inactivation of the c-MYC oncogene triggers tumor regression in T-cell acute lymphoblastic leukemia (T-ALL) resulting in dramatic changes to the chromatin landscape including DNA methylation. In this study, I investigated how MYC regulates DNA methylation and hydroxymethylation patterns to contribute to gene expression programs important for tumor maintenance in T-ALL and Burkitt lymphoma. I report that MYC maintains 5-methylcytosine (5mC) and 5-hydroxy-methylcytosine (5hmC) patterns by regulating the DNA methylation machinery, which is important for gene expression in T-ALL. DNA methyltransferases (DNMTs) initiate 5mC marks, while Ten-eleven translocation methylcytosine dioxygenases (TETs) oxidize 5mC to produce 5hmC as an intermediate modification, ultimately leading to active DNA de-methylation. I demonstrated that DNMT1 and DNMT3B are MYC target genes and that their expression is dependent on high MYC levels. Knockdown of DNMT3B in T-ALL reduced cell proliferation through cell cycle arrest and caused the reactivation of gene transcription through reversing promoter/CpG island methylation. Furthermore, I demonstrated that TET1 and TET2 expression is MYC-dependent, as high TET1 and low TET2 levels depend on oncogenic MYC. Knockdown of TET1 in T-ALL reduced cell proliferation through cell cycle arrest and caused genome-wide changes in 5mC and 5hmC corresponding to changes in gene programs important for ribosomal biosynthesis and protein synthesis. In contrast, ectopic expression of TET2 reduced tumor cell proliferation through apoptosis/necrosis and caused genome-wide changes in 5mC and 5hmC corresponding to changes in transcriptional regulatory gene programs. My finding that a coordinated interplay between components of the DNA methylation machinery is necessary for MYC-driven tumor maintenance highlights the potential of targeting specific DNMT or TET proteins for therapeutic strategies.

    Davila, Alec Christopher; Biomedical Sciences (Augusta University, 2019-05)
    Background. Heart failure with preserved ejection fraction (HFpEF) is often manifested as impaired cardiac and microvascular reserve, for which no current effective therapies are available. We sought to determine if conducted vasodilation, which coordinates microvascular resistance longitudinally becomes compromised in HFpEF. We tested the hypothesis that inhibition of adenosine kinase (ADK), the major adenosine-metabolizing enzyme and novel therapeutic target, augments conducted vasodilation; therefore, improving tissue perfusion and left ventricle (LV) diastolic function. Methods and Results. Conducted vasodilation was assessed ex vivo in coronary arterioles isolated from right atrial appendages of patients with or without HFpEF diagnosis and in skeletal muscle arteries of the rodent model of HFpEF, ZSF1 rats. Obese ZSF1 rats displayed LV diastolic dysfunction over a 20-week lifespan as indicated by reduced E/A ratio and increased deceleration time of mitral flow velocity observed on echocardiogram. Conducted vasodilation in both HFpEF patients and obese ZSF1 rats were significantly reduced, which was associated with increased vascular expression of ADK. Isolated arterioles incubated with ADK inhibitor, ABT-702 (0.1 μM) displayed an improved conducted vasodilation. In vivo treatment of obese ZSF1 rats with ABT-702 (1.5 mg/kg, i.p. for 8-week) prevented LV diastolic dysfunction, and in a crossover design, ADK inhibition improved conducted vasodilation and LV diastolic function. Furthermore, ABT-702 treatment reduced surrogate markers of myocardial hypoxia (carbonic anhydrase 9 expression and fibrosis) in obese ZSF1 rats. Moreover, mice with endothelium-specific deletion of ADK exhibited augmented vasodilation and were protected against the development of transverse aortic constriction-induced LV dysfunction. Conclusion. Collectively, upregulation of microvascular ADK impairs conducted vasodilation in HFpEF. Pharmacological inhibition of ADK improves microvascular vasodilator function and provides beneficial effects on myocardial perfusion and LV diastolic function in HFpEF.
  • Increased Membrane Thiol Oxidation in Sickle Erythrocytes

    Hill, Benjamin Albert; Department of Cell and Molecular Biology (1988-06)
  • Opioid Crisis Trends in Georgia: Using Data Management Systems to Better Inform Public Policy

    Sheikh, Nafiz; Tauhidul, Liniya; Medical College of Georgia
    Introduction: The nationwide opioid epidemic is arguably the most consequential public health crisis of the new millennium. Unfortunately, a dearth of medical literature exists analyzing the scope of the epidemic in Georgia. This presentation will investigate trends in fatal opioid overdoses in Georgia using a robust healthcare data management system: Center for Disease Control’s (CDC) WONDER. Methods: Using CDC WONDER, a cohort of all fatal opioid overdoses in Georgia from 1999 – 2017 was obtained (N=10,070). The group was then stratified by race, sex, age group, and overdosed opioid type. Time series analyses were used to determine trends, two-sided Chi-square tests with statistical significance set to p<0.05 used to compare opioid mortality proportions from different years and mortalities between age groups. Lastly findings were correlated to geography to ascertain if urbanization correlated to opioid mortality. Results: Approximately 1056 fatal opioid overdoses occurred in 2017, up 192% from 550 deaths in 2010. Fatal overdoses from heroin and synthetic accounted for only 2% and 17% of total deaths in 2010 but magnified to 20% and 32% by 2017 (p<0.05). Beginning 2013, heroin and synthetic opioids such as fentanyl together drove Georgia opioid mortality sharply higher. Among different age groups, Georgians aged 25-34yrs experienced the highest mortalities compared to other females within the same age group (p<0.05) and to males and female in the 35-44yrs and 45-54yrs groups (p<0.05). Correlating fatalities to geography found urban areas in Atlanta, Augusta, and Columbus to have the highest mortality rates. Conclusion: Georgians have experienced an unprecedented surge in mortality from opioid-related overdoses in recent years. With robust healthcare data management systems, however, new research endeavors are poised to generate more thorough epidemiological reports that will better inform local and state health policy.
  • Non-invasive Biomarkers to Detect Acute Kidney Injury in Premature Infants

    Marin, Terri; Williams, Bryan; Bhatia, Jatinda; Sharma, Ashok; Mundy, Cynthia; Cockfield, Christy; College of Nursing; Department of Pediatrics: Neonatology; Department of Population Health Science; Department of Obstetrics and Gynecology; et al.

    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.

    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.

    Dixon, Rachel; Mandavilli, Rohan; Department of Phychological Sciences; Department of Biological Sciences; Departmetn of Pharmacology & Toxicology; Bunting, Kristopher M; Alexander, Khadijah; Vazdarjanova, Almira; Augusta University (2019-02-13)
    Post-traumatic stress disorder (PTSD) is a psychological disorder that can occur after a traumatic event. Individuals with PTSD exhibit extreme anxiety and learning and memory difficulties. Once exposed, 12-35% develop PTSD with women twice as likely to be affected than men. Our goal is to discover underlying mechanisms to prevent PTSD, as we investigate the relevance of glutamic acid decarboxylase positive (GAD+) neurons on susceptible (SUS) and resilient (RES) male rats. SUS and RES phenotypes were assessed using the highly advanced RISP protocol to reveal susceptibility to a PTSD-like phenotype. The increase of GAD+ cells in the medial prefrontal cortex (mPFC) informs us that more GABAergic neurons are present, which can cause inappropriate recall. We will be examining if there is a difference in the number of GAD+ cells in the RES versus SUS male rats. To investigate, we used cryosectioned brains from SUS or RES rats. The brains were stained using immunohistochemistry to isolate the GAD+ neurons in the mPFC and were counted. The results of this experiment will be determined and examined at a later date closer to our presentation. We expect to see a SUS male rats to have a higher number of GAD+ neurons.

    Patel, Shrey P; Department of Phychological Sciences; Department of Pharmacology & Toxicology; Lambert, Nevin; Augusta University (2019-02-13)
    The experiment discusses the role of inverse agonist binding to receptors and how its effect cell signaling. The specific receptors that was focused on in the project was histamine receptor H1 (HRH1) and histamine receptor H2 (HRH2) which are types of G-protein coupled receptors (GPCR). Both receptors are activated when a ligand, specifically a histamine molecule, which binds to the receptor and activates the signaling pathway within the cell. The main protein within the signaling pathway is the G-protein which helps the cascade effect of the signal to other molecules. G-proteins are activated through GTP. An inverse agonist works like an agonist but will have an opposite end effect within the cell. It was originally thought that inverse agonist works the same way as an agonist to recruit a GTP and activate a G-protein for signaling. The experiment being tests tries to explain the opposite that the inverse agonist could activate the protein without GTP and continue to have its effect on the cell. Human embryonic cells were transfected with plasmids that contain sequences for the receptors and the G-protein, which were also tagged with a fluorophore to measure any bioluminescence with interaction of G-protein and the receptor when the ligands binds. From collecting data from the bioluminescence effect, it shows that there is an interaction a receptor and G-protein complex when the inverse agonist is bound.

    Vick, Sarah; Department of Biological Sciences; Department of Cellular Biology and Anatomy; Lu, Xiaowen; Watsky, Mitchell; Augusta University (2019-02-13)
    It is estimated that 41.6% of the US population suffers from vitamin D deficiency, with Blacks (82.1%) and Hispanics (69.2%) at even greater risk�Vitamin D deficiency can be caused by a variety of sources and given the wide range of causes, it is important to understand what measures this population might take to proactively prevent greater harm, or to reverse harm that might have already occurred. This project is designed to test the general hypothesis that Vitamin D deficiency exacerbates preexisting primary corneal pathologies. Previous research has established that the corneal epithelium in diabetic mice heals at a faster rate than the epithelium in diabetic vitamin D receptor (VDR) knockout (KO) mice. It is known that within diabetic mice, the corneal nerve density is decreased. However, it is unknown how VDR KO mice or vitamin D deficient with diabetes will affect corneal nerve density. In order to identify variabilities within the nerves that indicate slow wound healing, the mouse corneas will be collected, stained for confocal microscope observation, and analyzed through image processing to determine nerve density.

    Rajpurohit, Shubhra; Department of Ophthalmology; Thounaojam, Menaka C; Jadeja, Ravirajsinh; Gutsaeva, Diana; Bartoli, Manuela; Augusta University (2019-02-13)
    Retinal neovascularization (RNV) is a potentially blinding condition characterized by the development of small, leaky, abnormal, blood vessels in the retina. This occurs as a consequence of retinal ischemia, which promotes the release of angiogenic factors such as vascular endothelial growth factor (VEGF). MicroRNAs (miRs) are non-coding RNA involved in post-transcriptional regulation of genes resulting in the blockade of their expression. MiRs are key players in a wide range of biological processes such as cell differentiation, neurogenesis, viral infection, immunity, and hypoxia. More specifically, microRNA-21 (miR-21) is upregulated in many pathological conditions including cancer, and cardiovascular disease. Previously, we have shown that microRNA-21 (miR-21) is a downstream effector of the transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) in retinal endothelial microvascular cells. Here, we will identify new therapeutic targets as well as diagnostic tools to prevent retinal neovascularization and, potentially, other ocular diseases. One well-known retinal angiostatic factor is pigmented epithelium-derived factor (PEDF). Increased miR-21 expression in the ischemic retina affects PEDF �gene� expression. Interestingly, miR-21 is known to inhibit the expression of peroxisome proliferator-activated receptor alpha (PPAR?). PPAR? is a transcription factor for PEDF; therefore, increased miR-21 level in the ischemic retina could lead to inhibition of PPR alpha expression and consequent inhibition of PEDF expression.

    Patel, Chandani; Patel, Reeya; College of Science and Mathematics; Department of Orthopedic Surgery; Fulzele, Sadanand; Augusta University (2019-02-13)
    MicroRNAs (miRNAs) have been known to play a key role in bone regulation. Some miRNAs have been observed to increase bone formation via osteoblast formation and others seem to be involved in bone resorption via osteoclast formation. In this study, we aim to observe which miRNA of those secreted by cells during a traumatic brain injury (TBI) are involved in bone formation or bone resorption. Our focus miRNAs were: miRNA-151, miRNA-6991, miRNA-27a, miRNA-92, and miRNA-1224. Using mouse bone marrow monocytes (BMCs), we have induced osteoclast formation by feeding media containing macrophage colony stimulating factor (M-CSF) as well as receptor activator of nuclear factor kappa-B ligand (RANK-L). After osteoclastogenesis, it has been observed via tartrate resistant acid phosphatase (TRAP) staining that miRNA-151 and miRNA-6991 have been up-regulated during osteoclast differentiation. Of the ones examined in our study, miRNA-27a, miRNA-92, and miRNA-1224 have shown an increase during osteoblast differentiation. The observations from this study can contribute insight for creating possible therapeutic methods for osteoporosis related diseases.
  • The Study of 5ht-1d and 5ht-1f Receptor Interactions with Mini G Proteins via Bret Analysis

    Trang, Amy; Department of Chemistry & Physics; Department of Pharmacology and Toxicology; Spencer, Angela; Lambert, Nevin; Augusta University (2019-02-13)
    G protein-coupled receptors (GPCRs) are receptors involved in signal transduction, a process for converting extracellular signals into internal messages to elicit a cellular response. Signal transduction pathways involve activating various G protein subtypes (Gs, Gi/o, Gq/11�and G12/13) which typically lead to second messenger production. Traditionally, second messenger concentration assays are used to identify GPCR coupling with G protein(s), but they are not efficient in profiling GPCRs since they compare the concentrations from different downstream signals. Instead, novel tools, such as Bioluminescence Resonance Energy Transfer (BRET) and mini G (mG) proteins, can be used to profile GPCRs. BRET is a technique that provides quantitative data when protein-protein interaction occurs and requires the proteins of interest to be fused with either a bioluminescent protein or fluorescent protein. In this study, we used mG proteins representing each G protein subtype to identify 5-hydroxytryptamine (5-HT; serotonin) receptor coupling upon serotonin stimulation. Through BRET assays, we determined that both the 5-HT1D�and 5-HT1F�receptors couple primarily with the mGsiand mGo�classes of mG proteins. This supports previous studies that these receptors couple to Gi/o�proteins and suggests that the use of mG proteins in BRET assays is an effective tool for GPCR profiling.
  • Unconventional Coupling of 5HT7 receptors to Gs heterotrimers

    Adams, Elizabeth; Department of Pharmacology and Toxicology; Lambert, Nevin; Augusta University (2019-02-13)
    GPCRs play a major role in cell signaling through their interactions with heterotrimeric G proteins. In conventional models of GPCR-G protein coupling, agonist binding promotes a conformational change within the receptor, which then associates with G proteins, facilitating the exchange of GDP for GTP. GTP-bound G proteins dissociate from the receptor and exert their effects on downstream signaling molecules. Previous studies suggest that serotonin 5HT7 receptors associate with Gs�heterotrimers prior to agonist binding, and that 5HT7-Gs�complexes dissociate after the G protein is activated. Here we study this unconventional mode of coupling using bioluminescence resonance energy transfer (BRET) between luciferase-tagged 5HT7 receptors and Gs�heterotrimers labeled with Venus. Our results confirm that 5HT7 receptors interact with inactive (GDP-bound) Gs�heterotrimers in the absence of an agonist, and that this interaction is stabilized by the inverse agonist methiothepin. Stimulation with the endogenous agonist serotonin (5HT) decreased BRET between 5HT7 receptors and Gs, indicating that the activation of the receptor leads to 5HT7-Gscomplex dissociation. Interestingly, Gs�activation was not required for complex dissociation. These results are consistent with the hypothesis that 5HT7 receptors couple to Gs�heterotrimers via an unconventional mechanism involving ligand-sensitive complexes of receptors and inactive Gs.

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