SIBS student - Emily Courtman
SIBS mentor - Namasivayam Ambalavanan M.D.
Changes in transforming growth factor- b signaling and activation with stretching of mouse lung epithelial cells

Background: Many premature infants are born with severe respiratory problems resulting in high morbidity and mortality. Mechanical ventilation is sometimes necessary to ensure adequate gas exchange; however, it can cause lung injury. Ventilator induced lung injury (VILI) is considered to be due to volutrauma, or excessive stretching of the lung. Transforming growth factor- b (TGF-b) is a key regulator of growth inhibition, cell function, and extracellular remodeling that is involved in lung development and injury repair. It is possible that excessive TGF-b induced by volutrauma mediates VILI.
Hypothesis: Increased stretching of mouse lung epithelial cells increases active and latent TGF-b, mRNA, and protein.
Methods: Mouse lung epithelial cells (MLE-12) were cultured at passage 12 in HITES media. MLE-12 were plated at 6x105 per well on 6 well matrix bonded ProNectin culture plates (Flexcell International) and grown until 80-90% confluent. The growth media was removed and replenished with new growth media. Cells were stretched at 5%, 10%, and 20% elongation for 6 hours using the Flexcell Tension System. Controls for each of the various stretching groups were also used. Conditioned media and cells were then removed for TGF-b protein (by western blot and ELISA) and mRNA analysis (by RT-PCR).

SIBS student - BreeOna Ebrecht
SIBS mentor - John Hartman
A yeast model for development of CF therapeutics

Cystic fibrosis (CF) is an autosomal recessive disorder resulting from mutations of the Cystic Fibrosis Transmembrane Regulator (CFTR) gene, which serves as an ABC (ATP-binding cassette) chloride channel located on the plasma membrane. While there are over one thousand different mutations associated with CFTR in CF patients, the predominant mutation, accounting for approximately 70% of defective alleles and occurring in nearly 90% of CF patients, is a phenylalanine (F) loss at the 508 position (ΔF508), which prevents the protein from correct folding and biogenesis. The misfolded CFTR- ΔF508 is recognized by a protein quality control system associated with ER, and degraded through a process known as ER-associated degradation (ERAD). As a result, F508del-CFTR fails to reach the cell membrane, leading to physiological complications in CF patients. However, the CF phenotype varies too significantly to be well explained conclusively by the mutations in CFTR. This suggests that there are extragenic genetic modifiers of CFTR-∆F biogenesis, which can be potential drug targets to correct the CF phenotype. To therapeutically correct the defects of misfolded CFTR- ΔF508 biogenesis, a panel of small molecular compounds are synthesized and tested in mammalian models and some of them displayed potential correction effect to the CFTR- ΔF protein. Unfortunately, the actual drug targets and potential molecular mechanisms that underlie the correction have not been fully revealed due to the fact that mammalian models are difficult and costly to study.

The yeast model, YOR1 (Yeast Oligomycin Resistance), has recently been used to characterize CF disease. Similar to CFTR, there is deletion of the ΔF508 equivalent residue, ΔF670, in the first nucleotide binding domain (NBD) of Yor1, rendering the Yor1 protein misfolding and biogenesis defects. As an ABC transporter, Yor1 works as a drug pump to extrude the growth inhibitory drug – oligomycin (mitochondrial poison) from yeast cells. Therefore, we can monitor the correction of Yor1-ΔF670 protein by measuring and quantifying the yeast cell growth in oligomycin.

With this model, we are able to test the panel of CF compounds and screen for their potential drug targets and related biological pathways in yeast, which shares highly conserved pathways with mammals. There are two aims for this project, 1) expose the panel of corrector compounds to the yeast cells expressing Yor1-ΔF protein to identify promising compounds that work well with the model, and 2) analyze selected compounds in a genome-wide collection of yeast strains, each of which contains a single gene deletion and a regulatable Yor1-ΔF allele, to investigate potential related gene-drug interactions. Anther widely used CFTR- ΔF correction method, low temperature, will also be tested with this yeast model. The ultimate goal of this project is to further understand the biology of the compounds/corrections that could potentially be used in CF therapeutics.

SIBS student - Joshua M. Freda
SIBS mentor - Robert A. Kesterson
Analysis of Hypothalamic Feeding Circuits in Response to Induced Loss of Primary Cilium in Adult Mice: ISH Characterization of NPY, POMC, AgRP, and MCH mRNA

Primary cilia occur on most cells of the mammalian system and contain vital biochemical pathways involved in the intraflagellar transport of protein complexes that contribute to the functioning of the cell. Ciliogenic genes, Tg737 and Kif3a, encode proteins necessary to maintain these pathways and their disruption in hypothalamic neurons is associated with changes in feeding behavior. Since Tg737 homozygous null mice are embryonic lethal, we used a model of conditional deletion of the loxP flanked Tg737 gene in adult animals (via activation of a tamixofen-inducible cre transgene). The resulting mouse models were analyzed in regards to three experimental conditions including the baseline condition, after obesity has set in (observed at a maximum of 64 days after tamoxifen injection), and after weight loss has been observed through a restrictive diet regimen. To unravel the underlying mechanisms by which loss of cilia lead to changes in feeding behavior, we will characterize neuroanatomical changes in gene expression of the well-established feeding circuits in the hypothalamus. We will use in situ histochemistry with radioactive antisense riboprobes of coronal sections of the hypothalamus to map mRNA levels of orexigenic (e.g. neuropeptide Y (NPY) and agouti related protein (AgRP) in the arcuate nucleus, and melanin concentrating hormone (MCH) in the lateral hypothalamus) and anorexigenic (e.g. proopiomelanocortin (POMC) in the arcuate nucleus) neuropeptide signals. Our overall hypothesis is that one or more orexigenic signals (NPY, AgRP, MCH) will be upregulated in the obese state (with POMC downregulated), and that these changes will be maintained in normal weight diet-restricted animals.

SIBS student - Rebecca Garrett
SIBS mentor - Boris Pasche
Novel Mechanisms for the Treatment of Breast Cancer: In Vitro and in Vivo Models

For illness currently without adequate treatment available, like insomnia, a therapy called low energy emission therapy (LEET) has been developed. This treatment through electromagnetic fields (EMFs) is thought to have application as cancer treatment. In a previous Brazil Phase I/II clinical trial, several patients experienced long term tumor responses (evaluated by regular CT scans or ultrasound with contrast). These specific, discrete frequencies are unique for each malignancy evaluated and appear to affect gene expression and mitotic division, inhibiting cellular dividing in vitro. Growth inhibition is observed only in cancerous cells, while normal epithelial cells are not inhibited, suggesting specificity of inhibition.

In vitro and in vivo studies are necessary prior to a US clinical trial, specifically a large Phase III randomized control study. Most importantly, in vitro and in vivo experiments aim to gain an understanding of the mechanism of action for this treatment approach For in vitro experiments, normal breast cell line MCF10A (as an example of normal epithelium exposed to EMFs) and cancer cell lines MCF7, SKBR3, and 231 will be used. Cells will be subjected to a 21 hour period of EMF exposure. Cells can then be harvested for gene expression assays and Western blots. Real time PCR will be done to assess changes in genes PLP2 and XLC2, which will be determined by using a PCR generated standard curve. Western blots will be used to assess differing levels of PLP2 protein between treatment/control groups and to show apoptotic influences.

Mice will be used as the in vivo test subject. T cell compromised mice (athymic nude mice) and Non Obese Diabetic SCID mice are purchased from Harlan labs and injected with breast cancer cells. As tumors grow, they will be measured and tumor volumes calculated. Using an sXv27 (System for eXposure in Vivo) machine, mice will be subjected to EMFs for longer exposures than the in vitro cells. The endpoint for these experiments is excessive tumor burden. Tumors in mice are measured M-F to observe change in volume. This type of treatment is a novel therapy which has yet to show severe side effects, a rarity in cancer treatments.

SIBS student - Meredith Hubbard
SIBS mentor - Alecia Gross
Determining the Presence and Localization of Septin2 and Tulp1 in Rod Cells

Visual perception relies on the proper function of highly specialized neurons that sense light known as rods and cones. The rod outer segment (ROS) functions as the light-sensing organelle within rod photoreceptors and consists of thousands of membranous disks stacked within the plasma membrane. These disks form as vesicles produced within the cell body and are transported to the outer segment via the connecting cilium. Mutations within regions of the photoreceptor protein rhodopsin interfere with vesicular trafficking, leading to rod cell degeneration and the blinding disease autosomal dominant retinitis pigmentosa.

Based in part on findings from our lab and in others the carboxy-terminus of rhodopsin has been identified as the targeting element important for the proper trafficking of these vesicles due to its interaction with trafficking proteins in the inner segment and connecting cilium regions of the cell. We hypothesize a two-step mechanism of rhodopsin trafficking: the transport of vesicles containing rhodopsin from the Golgi apparatus in the inner segment through the connecting cilium to the outer segment and the assembly of these vesicles into disks at the base of the outer segment. While some of the proteins interacting with rhodopsin through both of these steps are known, some remain unknown and therefore the complete trafficking mechanism of rhodopsin bound vesicles and their assembly into disks is not completely understood. This project aims to determine the potential role of two known proteins, Septin2 and Tulp1, in the interaction with rhodopsin and trafficking of vesicles in both Mus musculus and Xenopus laevis. Presence of these proteins will be determined using Western Blot analysis and localization determined with Immunohistochemistry of retinal sections from both animals.

SIBS student – Andrea Loes
SIBS mentor – Michael Niederweis
Characterization of the periplasmic ferric binding protein of Mycobacterium smegmatis

Iron is an essential element for the growth of nearly all organisms. Bacteria have developed various acquisition systems to utilize iron in the environment. Here, we present the properties of MS3636, the periplasmic ferric binding protein of Mycobacterium smegmatis, a soil-dwelling, non-pathogenic microorganism often used in the study of Mycobacterium tuberculosis, the causative agent of T.B. It has been demonstrated that M. smegmatis has a low affinity ferric uptake system in which the inner membrane components display homology to the ferric uptake system of Haemophilis influenzae. In this study, we performed site directed mutagenesis on the periplasmic ferric binding protein MS3636 to elucidate amino acid residues necessary for iron binding. Mutant and wild type proteins were expressed in Escherichia coli and purified for comparison of iron binding. Iron binding was assessed by colorimetric assay and secondary structural analysis was performed with circular dichroism to verify proper folding of mutant proteins.

SIBS student - QueenDenise Okeke
SIBS mentor - Trygve O. Tollefsbol
The Effects of Green Tea Component, EGCG, on Triple Negative Breast Cancer Cells

Statistical studies conducted by The American Cancer Society have shown that when compared to Caucasian (CAU), Native American, Asian, and Latin American women, African American (AA) women tend to have lower occurrences yet a higher mortality rate of breast cancer. African American women who develop breast cancer frequently have estrogen receptor-α negative (ERα-neg) tumors which makes cancer treatment more challenging. ERα-neg breast cancers have a poor prognosis because they do not respond to current hormone-targeted therapies which target the ERα pathway. However, studies have shown that (-)-epigallocatechin-3-gallate (EGCG), a polyphenol found in green tea, can prevent and inhibit various types of carcinomas. In addition, EGCG can reactivate ERα expression in ERα-neg breast cancer cells. We hypothesize that breast cancer cell lines derived from AA and CAU women show differential responses to EGCG treatment. To investigate this, breast cancer cell lines will be studied to determine the changes in cancer-related genes after treatment with EGCG. Cell lines will be treated with various concentrations of EGCG for three consecutive days. The cells will be collected, analyzed, and viability will be determined via 3-[4, 5-Dimethylthiazol-2-Yl]-2, 5-Diphenyltetrazolium Bromide (MTT) assay. Additionally, morphological differences will be assessed and changes in mRNA and protein expression will be detected via reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis. Our findings reveal that EGCG can restore ERα expression, and this effect is enhanced when optimal dosages of EGCG are used. These results could help provide innovative and natural therapeutic applications for targeting the ERα gene in ERα-neg breast tumors of cancer patients using natural dietary ingredients like EGCG. Future studies, will be conducted to determine the long-term effects of EGCG treatment on breast cancer cells. Furthermore, natural dietary ingredients like EGCG may be used in addition with current chemotherapy agents to increase responsiveness of hormone-targeted therapies.

SIBS student – Jeremy Shepard
SIBS mentor – Joel Berry
Engineered polycaprolactone and cellulose substrates for MCF-7 F-108 co-cultures

Several studies have used 3D cell volumes to study established breast cancer cell lines. This project was designed to develop a co-culture of MCF-7 breast cancer cells and F-108 primary breast fibroblasts in a 3D volume on a biocompatible substrate. The MCF-7 cell line was engineered to express Vascular Endothelial Growth Factor (VEGF) from a doxycycline-inducible promoter and β-galactosidase constitutively. MCF-7 xenografts in mice treated with doxycycline have been shown to develop highly vascularized, desmoplastic, tamoxifen-resistant tumors which metastasize to the lungs. MCF-7 xenografts without doxycycline treatment grew into significantly smaller, non-metastatic tumors which responded well to tamoxifen. To replicate the behavior observed in vivo as recorded in existing literature, co-cultures were grown on polycaprolactone and cellulose scaffolds. Porous polycaprolactone (PCL) fibers were manufactured via electrospinning with salt leaching. Bacteria-grown cellulose disks were provided through collaboration with Chalmers University of Technology, Gothenburg, Sweden. A time course was run on cell co-cultures seeded onto each material with four treatments; with both, each, and without doxycycline and tamoxifen. MCF-7 cell proliferation was quantified using a β-galactosidase enzyme assay. Cell counts were used to determine whether or not the cultures exhibit increased proliferation and tamoxifen resistance under VEGF expression. This project serves two main purposes. The first is to have a functional model system for studying the role of Notch-1 signaling in VEGF-induced MCF-7 antiestrogen resistance. It is hypothesized that stromal cells produce the Notch ligand DLL-4 in response to VEGF, and that it binds to Notch-1 receptors on MCF-7 cells, actuating Notch signaling and inducing cell proliferation. The second is to establish the utility of electrospun polycaprolactone as a substrate for growing cancer cells for study in vitro. Further imaging may help to elucidate how MCF-7 and F-108 cells aggregate and grow into their culture substrates.

SIBS student - Aneesh Tyle
SIBS mentor - Ilan Kerman
5-Hydroxytryptamine 1B Receptor (5-HT1B) expression in the human caudal brainstem in major depression

Major Depressive Disorder (MDD) is one of the most common and harmful psychiatric illnesses in the US. Approximately 13% of Americans suffer from MDD during their lifetime. A great deal of depression-related research focuses on treating the cognitive and affective (or emotional) symptoms of MDD patients, as well as understanding the underlying brain dysfunction that causes these symptoms. However, depressed patients also frequently suffer other types of symptoms, such as chronic pain, cardiovascular problems, and motor difficulties. Our current research project aims to investigate the brain neural circuitry that may contribute to these more “physical” symptoms of depression.

There is abundant evidence of serotonin (5-HT) dysfunction having a major role in MDD. First and foremost - one of the most effective pharmacotherapies for MDD is to treat patients with medications that increase 5-HT function in the brain. Furthermore, previous human post-mortem studies have indicated that the organization of 5-HTergic neurons in the midbrain is altered in depressed patients. This work also revealed altered expression of certain genes involved in 5-HT neurotransmission (such as the gene tryptophan hydroxylase 2 (TPH2), a key enzyme required for 5-HT synthesis) in certain brainstem nuclei of MDD patients.

The present study focuses on the 5-HT1B receptor, which is an autoreceptor expressed on 5-HT neurons and has the capacity to regulate 5-HT neuron functioning. In this study, we used brainstem tissue collected from human subjects either psychiatrically normal or diagnosed with MDD. The brainstem tissue was cryostat sectioned and processed for in situ hybridization, which utilizes a radioactive probe to detect the expression of 5-HT1B receptor mRNA. My role in the project was to quantify the 5-HT1B receptor mRNA expression by measuring mean signal and integrated optical density from audioradiograms that were exposed to the radioactive slides labeled for 5-HT1B receptor mRNA. My analysis focused on these specific brainstem regions: gicantocellular pars alpha nucleus, ventrolateral medulla, raphe magnus, raphe interpositus nucleus, raphe obscurus, and the paramedian raphe nucleus. We focused on these regions since a previous study revealed TPH2 mRNA expression abnormalities in these areas in MDD. Based on those earlier findings, we hypothesize that 5-HT1B mRNA expression will also differ between MDD patients and controls in one or more of these brain areas.

SIBS Student: Daniel Bolus
SIBS Mentor: Raj Soorappan (rajnsr@uab.edu)
Tentative Title: Effect of Heat-Shock Stress on Cell Proliferation and Differentiation of in vitro Myoblast𝑪𝟐𝑪𝟏𝟐 Cells

Introduction: Myoblast C2C12cells are a unique line of muscle cells that can easily proliferate and differentiate. These cells are therefore a natural choice when studying effects of various environmental factors, in this case heat shock, on the differentiation of cells in vitro. This research project in particular will attempt to examine the effect of heat shock on these myoblast cells by determining its effects on cell proliferation, differentiation, and its induction of certain heat shock proteins that can serve to aid the myoblast cells in their reaction to stress.

Backgroundand Research Questions: Heat shock proteins, more accurately known as stress proteins, consist of a wide variety of proteins that usuallyserve as chaperones to protect other proteins in cells undergoing stress. In this research, this stressis caused by heat shock. These heat shock proteins, once activated, can also aid in future stress conditions. Some questions to consider include how much heat shock these cells can withstand before dying, and if heat shock proteins can actually benefit these myoblast cells.

Hypothesis: By inducing the activation of heat shock proteins, a certain amount of heat shock stress benefits the proliferation and differentiation of myoblast C2C12cells in vitro.

Methodology: The research plan is first to learn how to grow the myoblast cells in cell culture. This process beginsby preparing culture medium and seeding the cells to 70-80% confluence on 18plates. Once the plates are ready (maintained at a constant 37°Cwhen not undergoing heat shock), the followingprocedure for heat shock will be followed:
Day 1:Heat Shock 9 plates for 60 minutes at43°C, setting aside the other 9plates as the control plates
Day 2: Analyze three plates that underwent Day 1 heat shock and shock remaining 6plates for 30 minutes at 43°C
Day 3: Analyze three plates that underwent Day 1 and Day 2 heat shock and shock remaining 3 plates for 30 minutes at 43°C
Day 4: Analyze the three plates that underwent all days of heat shock Once these steps are learned, the heat shock process will be repeated for the proliferation and differentiation of the myoblast cells, following the procedure Dr. Raj outlined in the experimental plan for the project.

Expectations: Heat stress induces the production of heat shock proteins that aid in cell proliferation and differentiation by protecting cytoskeletal structures and enzymes.

Extensions: If the above research plan goes by quickly and efficiently, this research project can be extended by answering other questions that come to mind when considering heat shock proteins. For example, can these proteins be activated in other temperature-related stress conditions, such as exposure to cold temperature shocks or different amounts of heat exposure? Also, could other environmentalfactors such as oxidative stress induce similar proteins?

Applications: Working long hours in the heat of the sun, cooking in a hot kitchen, and even taking hot showers expose all of us to a lot of heat shock in our daily lives. Understanding our body’sreactions—good and bad—to this heat shock is key for something as simple as offering advice for proper exposure to this heat. This understanding also has applications to better understand diseases such as those that can lead to muscle atrophy, as it has been found that heat shock proteins could play a key role in maintaining muscle strength as we age.

SIBS Student: Clanton
SIBS Mentor: Sunnie Thompson (sunnie@uab.edu)
The Role of Non-Canonical Translation in Proliferation and Survival of Triple-Negative Breast Cancer

Metastasis to the bone is one of the most common causes of death in breast and prostate cancer. In order to survive and proliferate after metastasis, cellsmust be able to overcome several stress factors, such as hypoxia, starvation, and apoptotic signals. In general, initiation of translation in eukaryotes occurs by a cap-dependent mechanism, in which the 7-methylguanosine cap of the mRNA(messenger RNA)bound by initiation factors recruits the 40S ribosomal subunit, along with several other translation factors. However, under conditions of cellular stress, such as starvation or hypoxia,cells globally down-regulate cap dependent translation to conserve energy,and an alternative method of translation initiationthat is cap-independent is used. Briefly,an IRES (internal ribosome entry site) in the 5’ UTR of the mRNArecruits the ribosome internally at the start codon or upstream of it. mRNAs that contain IRESs code forproteins needed for survival and proliferation. Because cellswithin a tumorare under cellular stress, understanding the role of translation initiation during the cellular stress response will provide insight into mechanisms of tumor survival. One ribosomal protein, eS25, has been shown to be required for cap-independent translation, but not cap-dependent translation. Knockdown of eS25 in cancer cells provides us with the opportunity for the first time to understand the role ofcap-independent translation during tumor development. We are proposing to characterize the cellular responseof a triple negative breast cancer cell line (MDA MB 231)when eS25 is knocked down or not using in vitro assays to examine motility, invasion, proliferation, and susceptibility to apopotosis. In addition, we will identify cellular mRNAs that are eS25 dependent. Together, these studies will reveal the molecular mechanisms that underlie the role of cap-independent translation initiation in cell survival.

SIBS Student: Katelyn French
SIBS Mentor: Elizabeth E. Brown (elbrown@uab.edu)

The enzyme Ca2+/calmodulin-dependent protein kinase II (CaMKII) is expressed in heart cells (cardiomyocytes) and is in involved in several biochemical cascades within the cell. CaMKII is usually activated by Ca2+-bound calmodulin (Ca-CaM), but more recently, evidence has shown that CaMKII can be also activated by reactive oxygen species (ROS). Not only that, but this oxidation-induced CaMKII activation is strongly implicated to be associated with apoptosis, sinus node dysfunction, heart injury, and arrhythmias. The reason for this effect is not yet fully understood. A cell model containing ion channels, excitation-contraction coupling, mitochondrial energetics, and ROS production and scavenging has already been published under my mentor’s research. During my research time here, I hope to come to understand this pre-existing model and incorporate CaMKII dynamics into the model in such a way as to fit published experimental data. More specifically, I aim to create a function that models the behavior of CaMKII under oxidative stress.

SIBS Student: Sami Hudaib SIBS Mentor: Yang Yang (yangyang@uab.edu)
The effect of myeloma cell-derived Runx2 in bone microenvironments on mice models.

Multiple Myeloma(MM)isa plasma cell malignancy inthe bone marrow.Symptomsof MMinclude hypercalcemiaand release of protein elements in the blood that adversely affect organ systems like the kidneys and heart. MMnot only laysdormant in the bone marrow, but also orchestratesother cells and tissues aroundit to create a suitable tumor microenvironment by secreting soluble factors and cell-cell contact with bone marrow cells.This secretion results in the host's surrounding tissue acting abnormally to the signals received,providing the ideal conditions for growth,and eventually leadsto the tumor metastasis. However,the molecular network responsible for progression and bone-metastasis remainsto be determined.

Runx2, a member of the runt-related gene family, is well-known for its essential regulatory roles in osteoblastogenesis and bone formation. Indeed, substantial evidence indicates that various cancer cells, including myeloma cells, also express Runx2. A role for cancer cell-derived Runx2 in tumor-to-bone metastasis of solid tumors has been demonstrated. However, the function and role of MM cell-derived Runx2 in MM progression and metastasis remainunknown. Recent work from Dr Yang’s laboratory demonstrated that the expression of Runx2 in MM cells promotes tumor growth and bone metastasis. However whether or how myeloma cell-derived Runx2 alters bone microenvironment remains unclear.

My role in the study is to determine the changes of bone marrow cells by MM-derived Runx2and also see what tissue is more preferredfor tumor growth. This will be carried out by transforming mouse 5TGM1MMcells with Runx2that wereeitherknocked-in (Runx2 k/in)orknocked-down (Runx2 k/d)while comparing it to the control transfected 5TGM1cells(control).The cells were injected in micemodels that weresensitiveto getting MM(C57Bl/KaLwRij)through means of injections intratibially or intravenously drip inthe tail vein. Bone sections were already collected but I will (1) determine the immune status in bone marrow by immunostaining with antibodies against the markers of immune inhibitory cells; these include Myeloid-derived suppressor cells, regulatory T-cells, andregulatoryB-cells; (2) evaluatethe bone marrow angiogenesis by staining bone sections withCD31antibody;and (3) count the number of bone marrow mesenchymal stem cells (these cells are reportedly support MM growth and bone metastasis). I will compare the above immunostaining results between control and Runx2 k/in groups, as well as between control and Runx2 k/d groups.This study will further understand the role of MM-derived Runx2 in alteration of bone marrow microenvironment and provide insight into MM pathology.

SIBS Student: Alexander Hedaya
SIBS Mentor: Mark Bevensee (bevensee@uab.edu)
The Na/Bicarbonate Cotransporter NBCe1 Regulates Presynaptic Intracellular pH and Modulates Synaptic Transmission in the Hippocampus.

pH is a potentregulator of cellular processesand neuronal excitability in the central nervous system (CNS). Na-Coupled Bicarbonate Transporters(NCBTs) that shuttle bicarbonate into cellsand function as acid extruders are principle mediators of brain pH. Such transport alters intracellular pH (pHi)and extracellular pH (pHo) in a reciprocal manner, and both changes modify neuronal activity. In general, increases in pHostimulate neuronal excitability, whereas decreases inhibit. The Na/HCO3cotransporter NBCe1 in astrocytes plays a particularly important role, and human mutations in the SLC4A4gene encoding NBCe1 cause neurological abnormalities. According to the conventional model, neuronal activity increases extracellular K+that depolarizes astrocytesand stimulates NBCe1. In a negative feedback manner, NBCe1-mediated HCO3–transport into astrocytes acidifies the synaptic cleft and dampens further neuronal activity. In agreement with this model, the Dobrunz lab in collaboration with the Bevensee lab found that NBCe1 inhibitors enhanced basal synaptic transmission in the CA1 region of the rat hippocampal slice elicited by low-frequency (0.1-Hz) stimulation of the Schaffer collateralaxons. Surprisinglyhowever, the opposite was observed at high-frequency (50-Hz) stimulationand long-term potentiation(LTP). We hypothesize that NBCe1-mediated HCO3–transport is necessary for elevating presynaptic pHiand promoting synaptic transmission at high-frequency stimulation. In the current study, we will use an established technique of injecting the pH-sensitive dye BCECF-AM into the Schaffer collateral region of rat hippocampal slices to allow incorporation into CA1 presynaptic termini, and then use confocal microscopy to measure pHiunder various conditions. We will test the hypotheses that i) NBC inhibitors (S0859 and an L3 inhibitory antibody) reduce presynaptic pHi, and ii) these inhibitors unmask a pronounced decrease in presynaptic pHiwith high-frequency stimulation. Opposite results are expected with an L4 stimulatory antibody. Results from this study will shed light on the importance of pHiin modulating synaptic transmission, reveal a new mode of regulation by NBCe1, and implicate potential new targets in the treatment of neurological diseases such as epilepsy.

SIBS Student: Daniel Kulman
SIBS Mentor: Christopher Willey (cwilley@uab.edu)
Identification and localization of Myristoylated alanine-rich C-kinase substratein the nucleus as a prognostic factor in Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a life-threating cancer characterized bypoor prognosiswith a mediansurvival at diagnosis of 14 months. Treatments for GBMinclude surgical resection, radiotherapy(RT), and first-line chemotherapeutic temozolomide(TMZ).Total surgical resection of GBM is often limited due to poorly defined borders with normal brain tissuewith mostGBM tumors becomingresistant to RTand TMZleading to highly invasive GBMphenotypes.Conversely, O-6-methlyguanine-DNA methyltransferase (MGMT), a DNA repair protein has been implicated as a prognostic factor in TMZand RT sensitivity.Methylated MGMT promoter silences MGMT expression in the nucleus and is characterized with improved tumorsensitivity to TMZ and RT.Our lab previously identified myristoylated alanine-rich C-kinase substrate (MARCKS) as a protein that can affect GBM cellgrowth and RT sensitivity while being associated with improved clinical outcome. Indeed, we haveshown that knocking down MARCKS expression inU251GBMcells(high endogenous MARCKS levels)promotes tumor cell growth andincreased resistance to radiationwhile overexpressing MARCKS in U87 GBM cells(low endogenous MARCKS levels) suppressed growth and promoted asenescent phenotype. MARCKS is well characterized in severalprotein interactions on the plasma membraneincluding sequestration of phosphatidyl inositol bisphosphate(PIP2) and protein kinase B(Akt)pathways; and MARCKSeffector domain (ED)is a substrate forprotein kinase C(PKC). Interestingly, we have identified a novel MARCKS nuclear localization in GBM cells. SeveralPKCproteins regulatenuclear localization of diacylglycerol kinase-DGK-) by phosphorylating the ED,which is a nuclear localization signal (NLS). The ED in MARCKS is similar to NLS of DGK-and may function as a NLS in MARCKS. Therefore we hypothesize that MARCKS may have direct interaction on DNA repair mechanisms.By expressingdoxycycline-induciblefunctional domain mutants ofMARCKS in U87cells, we will characterize the location/distribution of mutant MARCKS protein in the cytoplasm and nucleus as well as determine MARCKS role in DNA repair processes.

SIBS Student: Sam LaMagna
SIBS Mentor: Alecia Gross (agross@uab.edu)

ABCA4 is a transmembrane protein located in the outer segment of rod photoreceptor cells responsible for transporting the chromophore all-trans-retinal out of the outer segment after its interaction with a photon of light and subsequent conversion from 11-cis-retinal. Mutations of the ABCA4gene cause Stargardt’s Disease, a form of macular degeneration, typified by deposits of yellow pigment, called lipofuscin, in retinal pigment epithelial cells, loss of central vision, and a delay in dark adaptation. Mutations also cause retinitis pigmentosa, a rod-cone dystrophy which results in progressive blindness. Thus, the structure of ABCA4 would be a very useful piece of informationin finding treatments for these diseases. However,it is unknown as of now. During my time in Dr. Alecia Gross’s lab in collaboration with Dr. Stephen Aller’s lab, I will be investigating what conditions are optimal for the purification, crystallization and Xx-ray diffraction of the ABCA4 protein to determine its strucuture. To do this, I will determine which conditions will give us the highest concentration, greatest activity, and greatest purity of ABCA4, so as to give us the greatest chance of ordered crystals in order to determine the protein structure using Xx-ray diffraction. Modified yeast cells from the Aller lab can heterologously express epitope tagged ABCA4, so to isolate ABCA4 from these eukaryotic cells, I will use a variety of detergents to solubilize the transmembrane proteins, purify them on an immunoaffinity column I will prepare, and perform ATPase ABCA4 protein assays to determine optimal conditions to give highly concentrated active protein to use in crystal trials. Should we obtain crystals, I will use the in-house X-ray beam in the Center for Biophysical Sciences and Engineering associated with the Aller labfor preliminary data collection.

SIBS Student: Marissa Luft
SIBS Mentor: Dabasish Chattopadhyay (debasish@uab.edu)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is an evolutionary conserved enzyme in the glycosidic pathway that catalyzes the phosphorylation of D-glyceraldehyde3-phosphate (D-G3H). The GAPDH enzyme of group B streptococcus (GBS) is known toincrease the bacteria’s virulenceand increase the production of the immunosuppressive cytokine IL-10 in host cells.The specific region of theGAPDH’samino acid sequence from 294-307 differs considerably from that of other organisms and protrudes from the protein. Additionally, it is known that this region has the ability to interact with plasminogen, actin, and fibrinogen, indicatingthatthis region could potentially enable the bacteria to bind to the host cell. Therefore, thisregion could beatarget for a possible vaccine. In order to produce an appropriate vaccine for GBS,it is important to better understand this enzyme, and take a closer look at the 294-307 amino acid regionand determine whether or not it is the immunodominate region of the protein. Protein crystallization and x-ray defraction will allow this region of the proteinand how it bindsto be visualized. This information willhelp determine the relationship between structure and functionof the protein. Additionally, western bloat technique can be used to understand the proteins interaction with antibodies. Finally, the structural and molecular differencesof this region in the GBS GAPDHindicate that antibodies targeting this region will not affect human GAPDH, but this hypothesis must be confirmed to ensure a vaccine targeting this region will not negatively effect host cells.

SIBS Student: Emily Milligan
SIBS Mentor: John Shacka (shacka@uab.edu)

Parkinson Disease (PD)is the second most common neurodegenerative diseaseaffecting 1% of Americans over age65. PD pathology is characterized by dopaminergic neuron loss and the accumulation ofinsolublealpha-synuclein (αsyn)aggregates known as Lewy bodies. Results of several pre-clinical studies suggest that αsynitself contributesto PD pathogenesis.Thus therapeutics aimed at promoting high capacity clearance of αsyn, in particular by enhancing function of the autophagy lysosome pathway (ALP),could be a valuable method for reducing αsyn pathology in PD.The goal of our lab is to target the lysosome as a strategy for promoting the autophagic clearance of αsynin PD. Our lab has recently published that αGalA-deficient mouse brains exhibit pathological accumulation of αsynconcomitant with ALP dysfunction. As an extension of these findings, we now have preliminary data indicating a significant reduction in αGalA enzymatic activity concomitant with an increase in αsynphosphorylated at serine 129 (P129Sαsyn), a pathological species that localizes to Lewy bodies in postmortem temporal cortex homogenates from late-stage PD patients compared to age-matched controls. Together these findings suggest that αGalA may attenuate αsynpathogenesis by promoting its lysosomal degradation in PD, but further studies are needed to validate this claim. We hypothesizethatincreasingαGalA promotes αsynclearance.For this study we will use two cells lines for in vitroanalysis. The SH-SY5Y line will be differentiated to a neuronal phenotype and used for evaluations of endogenous αsyn. The M17 cell line will be treated with Doxycyclin (Dox) for evaluation of clearance of over-expressed αsyn. Increased αGalA levels will be achieved by administration of Fabrazyme, a gene therapy drug used to increase αGalA levels in body cells of Fabry (a lysosomal storage disorder) patients.We will determinevia western blot analysis if transientover-expression ofαGalAby administration of Fabrazyme reduces basal levels of endogenous αsyn or increased levels by conditional over-expression. The experiment will be completedin differentiated SH-SY5Y human neuroblastoma cells, or αsynconditionally over-expressing M17 cells.

SIBS Student: Megan Moore
SIBS Mentor: Trent Tipple (ttipple@uab.edu)
ATG Dose Responses in Type II Murine Epithelial Cells

Bronchopulomary dysplasia (BPD) is the most commoncause of respiratory morbidity in premature infants.Infants make the transition from the intrauterine environment of approximately 3% O2to the extrauterine environment of 21% O2. When infantsare born prematurely,the lungs are notyet fully developedandthey are more susceptible to developing BPD. In order to maintain adequate blood oxygen levels, these babies are treated with highconcentrations ofoxygen(hyperoxia).Whenthe developing lung isexposed to hyperoxia,oxidative stress contributes to the developmentof BPD. However, the mechanismsby which oxidative stress contributesto BPD arenot fully understood. Data from our lab usingamurine model of BPD indicatesthata single doseofaurothioglucose (ATG), a gold-containing anti-inflammatory compound, attenuates hyperoxiclung injuryin newborn miceby enhancing antioxidant responses.Transcription of many antioxidant genes is regulated by nuclear factor E2-related factor 2 (Nrf2)andATG activates Nrf2-dependent gene transcription.Our data suggeststhat theeffects of ATG on Nrf2activation and subsequent antioxidant responses are mediated via inhibition of the redox enzyme thioredoxin (Trx) reductase. Other redox enzymes, including those of the glutathione (GSH) system, are also sensitive to inhibition by ATG. Alveolar epithelial cells are important for normal lung development.We hypothesize that ATG treatment will inhibit the Trx systembut not the GSH system, willactivate Nrf2-dependent gene transcription,and will protect MLE-12 cells from the deleterious effects of hyperoxia in a dose-dependent manner. Thesestudiesaresignificant because they willprovideimportant pharmacodynamicdata intoprotective effects of ATGin vitro. Theresults will aid in the interpretation of our in vivo findings in the murine BPD model by increasing our understanding of molecular pathways affected by ATG in lung epithelial cells. This project will support the potential therapeutic use of ATG to lessen the impact ofthe impact of oxygentoxicity in premature infants.

SIBS Student: Jason Moraczewski
SIBS Mentor: Glenn Rowe (gcrowe@uab.edu)
PDLIM3 a Regulator of Glycolytic and Oxidative Skeletal Muscle

Depending on which formof exercise one performs, one of twotypesof skeletalmuscleare involved. TypeI muscle fibers (slow twitch) are mainly used during lower-intensityexercises and depend on oxidative phosphorylation to fulfill the metabolic needs of the muscle. In contrast, Type II muscle fibers (fast twitch)are mainly used duringshort periods of high-intensity exercise and rely on glycolysis for energy.When an organismis performing prolonged aerobic exercise, the organism’s body will switchfrom using glycolytic to oxidative muscle fibers in order to maintain homeostasisand fulfillitsbody’smetabolic needs.The factors that play a role in this metabolic switching are not fully understood. Using a screen comparingoxidative versus glycolytic muscle gene expressionpatterns, we have identified PDLIM3 as a possible contributor to this metabolic switch. PDZ and LIM Domain 3 (PDLIM3) is found to be highlyexpressed inskeletal musclecompared to other tissues.The encoded protein from this gene has been shown to bind to spectrin-like repeats of alpha-actinin-2 and to colocalizewith alpha-actinin-2 at the Z lines ofsarcomeres.However, the role of PDLIM3 in fiber type switching is currently unknown. In order to further elucidate the metabolic role of PDLIM3in skeletal muscle, quantitative gene expression will be performed to determine the levels of PDLIM3 expression under various exercise and exercise-like conditions. We predict levels of PDLIM3 expression to be downregulatedunder aerobic exercise conditionsand this might be responsible for the metabolic switch seen in skeletal muscle in response to exercise. This researchproject aimsto outlinethe role of PDLIM3 and its significanceon metaboliccontrolin skeletal musclein response to exercise.

SIBS Student: Dana Pham-Hua
SIBS Mentor: Hubert Tse (htse@uab.edu)
TA/PVPON-containing nanothin coatings can suppress pro-inflammatory chemokine synthesis and potentially elicit islet transplant survival

Type 1 diabetes (T1D) is a chronic pro-inflammatory autoimmune disease consisting of reactive oxygen species (ROS), cytokines, and islet-infiltrating leukocytes involved in pancreatic β-cell lysis. One promising treatment for T1D is islet transplantation; however, its clinical application is constrained due to limited islet availability, adverse effects of immunosuppressants on islet function, and declining graft survival. Islet encapsulation may provide an immunoprotective barrier to help preserve islet function and prevent immune-mediated rejection after transplantation into T1D patients. We previously demonstrated that a novel cytoprotective nanothin coating for islet encapsulation consisting of tannic acid (TA), an immunomodulatory antioxidant, and poly (N-vinylpyrrolidone)(PV/PON), was efficacious in dampening innate and adaptive immune responses involved in transplant rejection. We hypothesize that in addition to suppressing pro-inflammatory cytokine synthesis, TA/PVPON will hinder the production of chemokines involved in recruiting immune cells to the site of islet engraftment. To test this hypothesis, bone marrow-derived macrophages were stimulated with poly(I:C) in the presence of TA/PVPON capsules to examine CCL2, CCL3, CCL4, CCL5, CCL17, and CXCL10 chemokine expression. TA/PVPON coatingseliciteda7-fold decreasein CXCL10, an important chemokine implicated in transplant rejection. ForCCL3 and CCL4, there was a 5-and 3-fold decrease in contrast to non-treated poly(I:C)-stimulated macrophages, respectively, providing additional evidence that TA/PVPON nanothin coatings could decrease leukocyte chemotaxis to sites of islet transplantation. Ourresults provide further supportthat TA/PVPON-containingencapsulated islets may suppress immune-mediated response to enhance islet allo-and xenograft acceptance to restore euglycemia in T1D patients.

SIBS Student: Ilya Pound
SIBS Mentor: John Hartman (jhartman@uab.edu)
Investigation of mechanistic differences between gemcitabine and cytarabine through analysis of gene-drug interactions in S. cerevisiae

While sharing similar structures, nucleoside analogs are a well-known class of cancer therapeutic drugs that differ in the types of tumors they treat. Two of these chemotherapy agents are gemcitabine, which is used to treat breast, ovarian, pancreatic and non-small-cell lung cancer, and cytarabine, which is used to treat a range of hematological cancers. Although both of these compounds inhibit DNA replication in rapidly dividing cells and have been in use for over 20 years, there is little explanation for why these drugs exert efficacy against such different types of cancer. A deeper understanding of the mechanisms by which the important nucleoside class of anti-cancer agents works could lead to discovery of more effective treatment regimens. It is difficult to develop genetics-based strategies for cancer treatment due to the complexity of multiple mutations, as well as the interaction of cancer genes and pathways. One strategy to shed light on these complicated mechanismsis the analysis of gene-drug interactions in the model organism S. cerevisiae,whereQuantitative High Throughput Cell Array Phenotyping (Q-HTCP) can be used to ascertain from a genomic collection of ~6000 yeast gene deletion strains, the role of every gene in drug resistance. To use this approach, we will transform the 6000 strains for expression of human deoxycytidine kinase (dCK), which sensitizes yeast to growth inhibition by nucleoside analogs. With the dCK-expressing collection we will quantify the interactions for all yeast genes with cytarabine and gemcitabine. The genes that result in synthetic lethality with nucleoside toxicity will represent new anti-cancer targets, which then can be studied in their respective homologs in humans. If we can define a network of genes that determine the cellular response to nucleoside drugs, we can couple our findings with cancer genome data to deduce why particular cancers are subject to nucleoside toxicity, while some are not. Understanding which mutations make yeast more susceptible to these drugs will provide hypotheses regarding mutations in cancer that sensitize them to these drugs. If successful, this approach could contribute a predictive model to test in the context of precision medicine and selecting anti-cancer therapy based on the genetic makeup of the target tumor.

SIBS Student: Summer Rhynes
SIBS Mentor: Colin Martin (Colin.Martin@childrensal.org)
The Role of CCL28 in Neonatal Intestinal Protection

Proper development of the neonatal immune system is critical to establish intestinal homeostasis and protection. Immunoglobulins secreted by developing B cells play a primary role in the process preventing translocation of harmful mucosal pathogens. In humans this process takes up to 2 weeks to develop and the factors that are necessary for process to incur are not well understood.B cells also act as cytokine producers and what type produced determines the immune response. Chemokines are a type of cytokine that play a role in intestinal lymphocyte localization and intestinal immunity by guiding different leukocyte subsets to a given destination. They are small, secreted chemotactic cytokines that control the innate and adaptive branches of the immune system.In addiction to their function in cell homing and migration, numerous chemokines have been found to exhibit direct antimicrobial activity against Gram-negative and Gram-positive bacteria. There are also certain chemokines play important roles in B cellfunction, specifically antibody production. A specific example is CCL28, a chemokine that directs the migration of IgA+ plasma cells to mucosal sites. Sites of human CCL28 expression are the salivary, bronchial, and mammary glands along with the small intestine and colon. Further investigation of CCL28 is ongoing because it could contribute to regulation of host mucosal defense at homeostasis as well as under infection or inflammation. The purpose of this experiment is to determine CCL28 expression in miceusing RT-PCR, in rat intestinal epithelial cell lines and its repose to bacterial stimulation, and in human neonates with NEC compared to controls immunohistochemistry. RT-PCRwill be usedin newborn, 7 weeks, 8 weeks, and 14-week-old mice. After interpreting how this chemokine is expressed, results will hopefully lead to a better understanding of the mechanism for its expression. With a full comprehension of the mechanism it can be manipulated, which could play an important role in antibody production andmigration.

SIBS Student: Cason Robbins
SIBS Mentor: Rita Cowell (rcowell@uab.edu)

Peroxisome proliferator-activated receptor coactivator-1α (PGC-1α) is a transcriptional coactivator that has been implicated in the pathophysiology of various neurological disorders. Its function in central nervous tissue has remained largely unknown until recently. PGC-1α has been observed to significantly regulate the expression of various proteins necessary for proper neural function and neurotransmitter release. Mice lacking PGC-1α in pyramidal neurons of the hippocampus and cortex exhibit hyperactivity and reductions in synaptotagmin II (Syt2) in the dentate gyrus of the hippocampus. Syt2 is believed to be involved in vesicular trafficking and is necessary for proper synaptic release of neurotransmitters.In previousexperiments, PGC-1α loss showed no significant effects on working memory. However, thedentate region of the hippocampus is involved in object location memory, which could potentially be affected by loss of PGC-1α in pyramidal neurons. In this experiment,mice aresubjected to a behavioral protocol that testsboth object location memory and novel object recognition to determine the effects of PGC-1α loss on these processes. We expect the loss of PGC-1α to negatively impact object location memory and novel object recognition in mice. This study will shed more light on the effects of PGC-1αin proper neural functioning, and it will hopefully lead to a better understanding of the functional impact of PGC-1α deficiency in neurological disorders.

SIBS Student: David Sams
SIBS Mentor: Martin Young (meyoung@uab.edu)
Regulation of Myocardial Fatty Acid Oxidation by the Cardiomyocyte Circadian Clock: Potential Role of Biotinylation

Background: Circadian clocks function as independent time-keeping devices within cells in the body that allow the body to be better prepared for the demands that face an organism at different points throughout the day. The circadian clock primarily functions through the heterodimerization of the transcriptional factors CLOCK and BMAL1. It has been shown that cellular metabolism is regulated by the circadian clock to account for varying energy demands placed on cells throughout a circadian cycle. As 70% of ATP necessary for contraction in the heart comes from fatty acid oxidation, it is critical to understand the role of the cardiomyocyte circadian clock in myocardial fatty acid oxidation. One of the main pathways for regulation of fatty of acid oxidation in the heart is modifying the activity of biotin-dependent acetyl-CoA carboxylase 2, which produces malonyl-CoA, a coenzyme that limits fatty acid oxidation. Previous studies have found that the genetic disruption of the cardiomyocyte circadian clock increases fatty acid oxidation while decreasing the biotinylation of acetyl-CoA carboxylase 2. Thus, we hypothesize that the cardiomyocyte circadian clock regulates myocardial fatty acid oxidation through the biotinylation of acetyl-CoA carboxylase.

Methods: Cardiomyocyte specific BMAL1 knockout mice and wild-type background mice will be used as models for a disrupted and a functional circadian clock, respectively. The role of biotin and biotinylation will be examined using four different groups of mice: a BMAL1 knockout group being fed a standard lab diet, a BMAL1 knockout group being fed a biotin enriched diet, a wild-type control group being fed a standard lab diet, and a wild-type group being fed a biotin enriched diet. Each group will receive their selected diet for six weeks. In order to measure the fatty acid oxidation in the heart, an ex-vivo working mouse heart perfusion will be performed on six mice from each group using a buffer with 3H-labeled oleate. The 3H-labeled water, the product of the beta-oxidation, will be separated out of the buffer using ion exchange chromatography and quantified using a liquid scintillation counter.

Anticipated Results: We predict that the BMAL1 knockout mice will have higher rates of fatty acid oxidation in comparison to the wild-type mice, as the disrupted cardiomyocyte circadian clock will prevent the biotinylation of acetyl-CoA carboxylase. Furthermore, we predict that the biotin enriched diet will normalize the biotinylation of acetyl-CoA carboxylase in the biotin diet BMAL1 knockout group, decreasing the amount of fatty acid oxidation in comparison to the standard diet BMAL1 knockout group.

SIBS Student: Austin Williams
SIBS Mentor: Rakesh Patel (rakeshp@uab.edu)
Investigation of Hemoglobin Degradation and Subsequent Heme ReleaseMechanisms by Oxidative Stress Using Spectral Deconvolution

Transfusion of stored red blood cells is a commonly usedtreatment for anemic trauma patients. FDA guidelines allow stored red blood cells to be used up to 42 days after the blood is drawn. However,increasesin morbidity and mortality, acute lung and kidney injuries, and pneumonia havebeen observed in these patientswho received older packed red blood cell units.During storage, red blood cells undergo several biochemical and morphological changes includinghemolysisresulting in free hemoglobin.This phenomenon, which iscommonly referred to as storage lesion, is thought to be the underlying cause of the adverse effects observed with the transfusion ofpacked red blood cellsthat are nearing their expiration date.Our group has recently shown that free heme is also formed during red cell storage and mediates post-transfusion toxicity in experimental models of trauma. This gives rise to the question, how is free heme released during red blood cell storage? We hypothesizethat oxidative stress leads to hemoglobin degradationand the release of heme. To test this experimentally,oxygenated hemoglobin, methemoglobin,hemolysate, and catalase-free hemoglobinweresubjected to varying concentrations of common oxidizing agents that are found in biology andspecificallyblood storage conditions. UV-Vis spectrophotometry pairedwithspectraldeconvolution wasused to determine the composition of the hemoglobin solutionsfollowing their exposure to variousoxidizing agentsincluding hydrogen peroxide, nitrite,andhypochlorous acid.It was found that physiological concentrations of hydrogen peroxide and nitrite did not significantly inducetherelease of free heme from oxygenated hemoglobin or hemolysate. However, a significant, dose-dependent release of free heme was observed following the administration of oxidizing agents to catalase-free hemoglobin. These data suggest that exposure to physiological levels of oxidants are unlikely to significantly mediate hemoglobin breakdown and heme release.

SIBS Student: Mashhood M. Wani
SIBS Mentor: Dan Gorelick (danielg@uab.edu)
Physiological estrogens exert opposing effects on heart rate in zebrafish embryos

Estrogens are a group of hormones that influence the development and function of diverse organ systems, such as the reproductive, nervous and cardiovascular systems.Acute exposure to 17β-estradiol (E2) increases heart rate in zebrafishembryos, an established model for cardiovascular development. However, the effect of acute exposure to other physiological estrogens on heart rate is unknown. Here we show that the physiological estrogens estrone (E1) and estriol (E3) have opposing effects on heart rate. 50-hour post fertilization zebrafish embryos treated with exogenous E1 had a decrease in heart rate while embryos treated with exogenous E3 had an increase in heart rate. Estrogens signal through two different receptors: nuclear estrogen receptors (ERα, ERβ) that are ligand-dependent transcription factors, or the G-protein coupled estrogen receptor (GPER),an integral membrane protein that activatessecondmessengersin the cytosol. E2 acts via GPER to increase heart rate, but whether E1 or E3 modulate heart rate via GPER is not known. To test whether E1 and E3act via GPER to change heart rate, we treated GPER mutant zebrafish with E1 and E3 for one hour and measured heart rate. We found that E1 led to a decrease in the heart rate while the E3 did not cause an increase in heart rate suggesting E1 acts in a GPER-independent mechanism while E3 acts in a GPER-dependent mechanism. Our results demonstrate that although estrone, 17β-estradioland estriolare structurally similar, each has its own unique downstream physiological effect on heart rate.

SIBS Student: James Ansell
SIBS Mentor: Palaniappan Sethu (psethu@uab.edu)
A Biomimetic Cardiac Tissue Model (BCTM) to determine effects of cellular orientation on structural remodeling and function

Myocardialinfarctions present one of the leading causes of death in America. The adverse effects of infarcted/damaged cardiac tissue results in the decline of patients’ health due,in part,to the hypertrophic responses of surrounding tissues as the hearts tries accommodating to a decrease of cardiac output. Cardiac patches, consisting of cardiac tissue grown in vitrohave been suggested as a novel treatment modality for infarcted patients. It has been shown that cardiomyocytes tend to orient themselves along primary axis of strain. However, if cardiac patch technology is employed,these cells may end up oriented in a matrix that forces these cells to be aligned against these primary strain profiles. This study evaluated the effects of orientation of H9C2 rat cardiomyoblastic cellsusing a novel biomimetic cardiac tissue model(BCTM). Cells cultured within the BCTM in anelliptical cell culture chamber experience stretch predominantly along the short axis of the ellipse. By patterning cells either in line with the primary direction of stretch or perpendicular to the primary direction of stretch, we sought to determine the effects of stretch on structural remodeling. Overall, we measured cell thickness, cell length and ratio of cell thickness to cell length to quantify the type of hypertrophy. Our results suggest that cells aligned parallel with the primary direction of stretch have thicker morphologies than cells aligned perpendicular to the direction of primary stretch.

SIBS Student: Ananya Bandyopadhyay
SIBS Mentor: Douglas Hurst (dhurst@uab.edu)
Regulation of SIN3A and SIN3B expression in metastatic breast cancer cells by unique microRNA

SIN3 chromatin remodeling complexes have been demonstrated to regulate breast cancer progression and metastasis. More recently, the Hurst lab has shown differential functions for the two paralogs of SIN3, SIN3A and SIN3B, with data supporting metastasis suppressive roles for SIN3A and metastasis promoting roles for SIN3B. Although SIN3A and SIN3B share a significant amount of sequence similarity, they are quite different within their 3’ untranslated region (UTR) leading us to hypothesize that SIN3A and SIN3B expression is regulated by unique microRNA (miRNA). We utilized three publicly available databases (TargetScan.org, miRDB.org, and microRNA.org) to obtain putative miRNA that target SIN3A and SIN3B. We identified 9 miRNAthat are predicted to target SIN3A, 5 miRNA for SIN3B, and4 miRNA forboth paralogs.Future studies willvalidate the direct binding of these putative miRNA to the 3’ UTR of SIN3A or SIN3B using luciferase reporter assays and test the ability of each validated miRNA to lower the expression level of SIN3A or SIN3B in human cell lines. A more complete understanding of how the expression these genes is uniquely regulated is an importantstep towards comprehendingtheir role in the process of breast cancer metastasis and may help elucidate possible targeting strategies for patients.

SIBS Student: Emma Bloom
SIBS Mentor: Rosalinda Roberts (rcusidor@uab.edu)
Immunohistochemical analysis of excitatory and inhibitory input to the substantia nigra in schizophrenia

Schizophreniais awidespread psychiatric disorder in which dopamine abnormalities have been implicated.Schizophrenic patients haveexcessive dopamine in the striatum,and antipsychotic drugs primarily block dopamine receptors. The focus of the presentstudy was on the substantia nigra (SN), a basal ganglia nucleus that hosts one of the largest clusters of dopaminergic cells in the brainand projects to the striatum. Previous research has demonstrated hyperexcitability in the SN of schizophrenicpatients, but the cause of this is unknown. We aimed to determine whether this abnormality is modulatedby increased excitatory (glutamatergic) input and/or decreased inhibitory (GABAergic) input. Postmortem SN ofschizophrenicpatients (SZ, n=6) were compared to matched controls (NC, n=5).We used immunohistochemistry to localize the vesicular glutamate transporters,vGLUT1 and vGLUT2, and the GABAprecursor,GAD67. Densitometry was performed to calculate the density of glutamatergic and GABAergic terminals in the SN of SZand NCsubjects. No significant differences were obtained for the density of vGLUT1(NC: mean=18.3±5.5; SZ: mean=12.6±6.2), vGLUT2(NC: mean=36.4±3.6; SZ: mean=41.8±6.4), or GAD67(NC: mean=26.3±13.6; SZ: mean=33.2±19.7).VGLUT2 trended in the anticipated direction, so we aim to draw conclusive results with a larger sample size. This study contains possible limitations, including a small cohort, confounds arising from the use of postmortem tissue,and our subjects’variableuse of antipsychotic drugs. Regardless, inbetterunderstanding the reason behindhyperactivity of dopaminergic neurons in the SNof schizophrenic patients, we will be better equipped to identify targets for new treatment mechanisms.

Keywords: schizophrenia, substantia nigra, dopamine, glutamate, GABA

SIBS Student: Blake Brown
SIBS Mentor: Candace Floyd (clfloyd@uab.edu)
Localizing the corticospinal and rubrospinal tracts within the porcine spinal cord

Introduction: Although spinal cord injuries (SCI) are a devastating clinical problem, there continues to be failure in translation of research. Rodents continue to be the main and most well established model for traumatic spinal cord injury research. However, motor tract anatomy differs between humans and rodents. Because of this, some research groups are turning to the pig as an intermediate model due to their extensive anatomical and physiological similarities to humans. However, the neural anatomy and localization of major spinal tracts of the pig spinal cord has not yet been described. The goal of this study is to localize the major motor tracts, the corticospinal tract (CST) and the rubrospinal tract (RST), within the pig spinal cord.

Methods: Mature female domesticpigs received injections of fluorescentdextrantracers into theprimary motor cortex and thered nucleus using StealthStation®image guided navigation. 5-6 weeks post injections, the pigs were euthanized and their brain tissue and spinal cord were collected. The tissue wasserially sectionedandexaminedusingconfocal microscopy to observe to location of the CST and RST.

Results: The results demonstrated that the CST in pigs is laterally located in the white matter, very similar to that of humans.However, the CST does not appear to descend past the cervical regions. The exact location of the RST cannot be determined at this time, but preliminary results also point to a lateral location within the white matter throughout the entire length of the neural axis.

Discussion: The location of the CST and RST in the porcine model areanatomically similar to humans. Further work needs to be done to pinpoint the exact location of the RST, however, these results point to the porcine model as a valuable pre-clinicaltoolto improve translation of promising SCI treatments.

Keywords:Porcine, Corticospinal, Rubrospinal, Spinal Cord Injury

SIBS Student: Abby Fyfe
SIBS Mentor: N. Ambalanannan (ambal@uab.edu)

Introduction:Preterm infants often require respiratory support including supplemental oxygen, exposing lungs to higher than normal oxygen concentrations (hyperoxia). This oxidative stress can harm lung cells and lead to chronic lung disease called bronchopulmonary dysplasia (BPD). The mechanisms by which oxygen induces injury are little understood at the cellular level. Our laboratory has become interested in how lung cells communicate with each other via release of small particles called exosomes, and how changes in these exosomes may mediate some of the effects of oxygen toxicity.

Methods:Human broncho-epithelial cells (HBE) were exposed to both hyperoxia and room air. Exosomes were isolated from the media supernatants in which the cells are grown. The exosomes were counted and analyzed for vascular endothelial growth factor (VEGFA), basic fibroblast growth factor (FGF-2), and three microRNAs involved in lung injury (miR214, miR219, and miR876).

Results:Hyperoxia exposure decreased VEGFA and FGF-2, increased miR214, and did not change miR219 compared to room air controls. miR876 was not undetected in exosomes. The overall number and size of the exosomes increased with hyperoxia.

Discussion:The alterations in exosomes induced by hyperoxia may mediate some of the effects of oxygen toxicity. The decrease in VEGFA and FGF-2 as well as increases in miR214 may impair lung development. The lack of change in miR219 and the lack of miR876 indicate that these miRNA probably do not affect the development of BPD via exosomes.

SIBS Student: Gerges, Ereny
SIBS Mentor: A. Brent Carter (bcarter1@uab.edu)
Cadmium from cigarette smoke increases the risk and severity of lower respiratory tract infection by decreasing the immune response of alveolar macrophages

Cigarette smoking is associated with an increased risk of lower respiratory tract infection (LRTI). LRTIs are a prevalent infection that result in hospitalization; however, the molecular pathogenesis of cigarette smoke-induced LRTIs is unknown. Cigarette smoke contains more than 4500 chemical compounds, including carcinogens, toxins, oxidants, and metals. Cadmium (Cd) is one of the metals present in cigarette smoke. Cd specifically inhibits bacterial clearance in alveolar macrophages unlike other immune cells. The molecular mechanism by which Cd alters the immune response of alveolar macrophages is not known. One factor that is required for the immune response is ROS production by the NADPH oxidase. The small GTP-binding protein Rac2 is required for NADPH complex formation. We hypothesize that Cd from cigarette smoke impairs the host defense of alveolar macrophages via inhibition of the Rac2 GTPase. We tested our hypothesis by treating macrophages with different concentrations of cigarette smoke extract (CSE)or Cd to determine if there is a dose-dependent effect on Rac2. We isolated the cell fractions after treatment and found that localization of Rac2 in the cell membrane and ROS production by the NADPH oxidase are reduced in a dose-dependent manner. The mechanism of Rac2 inhibition is by impairing lipidation of the C-terminal cysteine. Overall, the results of these studies will allow us to characterize the molecular mechanism(s) by which cadmium in cigarette smoke inhibits host defense, thereby permitting the potential design of a clinically relevant therapeutic agent in subjects that smoke.

Keywords: Lower Respiratory tract infections (LRTIs), Rac2, Cadmium, Cigarette Smoke, Host defense of Alveolar Macrophagesand NADPH Oxidase.

SIBS Student: Lee, David
SIBS Mentor: Palaniappan Sethu (psethu@uab.edu)

The use of cell culture and animal models in biomedical research is prevalent, especially in the development of pharmaceuticals. However, these models present inherent limitationsincluding poor translation in clinical trials:static cell culturemodelsmay not replicate the environment observed in vivohowever, these models can be constructed with human cells but inorder for these models to be effective, critical aspects of the in-vivo environment need to be recreated.. Recent studies havereplicated various systems in the body, such as the alveolar-capillary interface where gas exchange occurs. Previous works have developedsmall scale devices that mimic this functional region, separating lung epithelial cells frommicrovascular endothelial cellsusing aporousmembrane ofpolydimethylsiloxane (PDMS). To improve upon previous designs, we constructeda devicewith a layer of fibrin gel, rather than porous PDMS,upon which a monolayer of cells were grown. Fibrin, a naturally occurring protein involved in blood clotting, has been utilized extensively as a cell scaffold because it more accurately mimics the extracellular matrix. Once cells have been cultured onto the fibrin membrane, the device will be placed in a closed loop with a steady flow of media withcontrolled amounts of pressure and shear stresseson the endothelial side and air on the epithelial side, mimicking the gas exchange architecture seen in vivo. This device will be further characterized by permeability assays using fluorescently labelled dextran to ensure that permeability of this device matches what is observed in the body.In summary, this model recreates the 3D architecture seen in the lung alveolus and will enable recreation of both normal and pathological alvelolar function for preclinical drug testing.

SIBS Student: Joshua Lee
SIBS Mentor: Mohammad Hassan (hassank@uab.edu)

Bone mass loss disorders,such as osteoporosis, affects over 53 million people in the United States (NIH Osteoporosis Overview). This issues a calling to develop novel and effective therapeutic approaches to treat this growing issue. Chromatin remodeling by the BAF complex plays a central role in the regulation of the differentiation of many cell types, including those that are involved in skeletogenesis. We have identified BAF45a, a component of the BAF complex, to play a major role in osteoblast differentiation. The purposeof this study is to define the role of BAF45a in osteoblast differentiation by studying osteoblasts’ gene expression profile during differentiation. Two main transcription factors that are involved in osteoblast differentiation that will be used in this study are RUNX2 and SP7 (Osterix). Real time PCR and Western Blottingwill be utilized in order to monitor these factors. By observing the levels of these factors and comparing them with the levels of BAF45a, some major conclusions could be drawn. The following marker genes will be used in order to track specific stages of cellular differentiation: COL1 (Collagen Type 1) as an early marker, ALP (Alkaline Phosphatase) as a marker of mature osteoblasts, and BGLAP (Osteocalcin) as a late marker of mature osteoblasts. A glycolysis enzyme, GAPDH, will be used as an internal control. The results showed that BAF45a exhibited a similar expression pattern to RUNX2. Since RUNX2 is the master transcription factor, this gives strong evidence that the BAF45a is crucial in the process of osteoblast differentiation.

SIBS Student: Luikart, Shelby
SIBS Mentor: Jeonga Kim (jakim@uab.edu)

Epigallocatechin-3-gallate (EGCG), a major component polyphenol in green tea, has been associated with the reduction in the symptoms of Type II diabetes through ECGC-induced autophagy in endothelial cells.1,2,3However, the dependence of autophagy on these metabolic effects remains inconclusive. The proposed experiments attempted to rectify this disparity by conducting various studies with diet-induced obese (DIO) mice with a deficiency in the endothelial specific autophagy gene 7 (ATG7), which is essential for macroautophagy.2Four groups of mice (control with or without EGCG, and endothelial-specific ATG7 KO [End-ATG7-KO] with or without EGCG) were exposed to a treatment regimen for 8-12 weeks. All mice received a 45% high-fat (HF) diet, but only two groups (one in the control, one in the KO) received daily gavages of EGCG. After treatment, their body weights were measured and their blood glucose levels were evaluated via the glucose (GTT), insulin (ITT), and pyruvate tolerance tests (PTT). The presence of P62, a protein that targets cells for autophagy,4was evaluated and quantified via Western blot. Overall EGCG showed similar effects on glucose levels for both wild type and ATG7 knockout mice as well as increased autophagy in iMAEC (Immortalized Mouse Aortic Endothelial Cell) lines. However, long-term treatment with EGCG had adverse effects on insulin tolerance and gluconeogenic activities in both WT and End-ATG7-KO mice. Thus, despite EGCG increasing autophagy, the physiological effects of EGCG on glucose homeostasis may be a detriment to the cell by affecting other metabolic pathways.

1.Kim, Jeong-a; Montagnani, Monica; Kon Koh, Kwang; Quon, Michael J. (2006). Reciprocal Relationships Between Insulin Resistanceand Endothelial Dysfunction. Circulation.113, 1888-1904.
2.Kim, Hae-Suk; Montana, Vedrana; Jang, Hyun-Ju; Parpura, Vladimir; Kim, Jeong-a (2013). Epigallocatechin Gallate (EGCG) Stimulates Autophagy in Vascular Endothelial Cells.J. Biol. Chem. 288, 22693-22705.
3.Jang,Hyun-Ju;D. Ridgeway, Simone; Kim, Jeong-a (2013).Effects of the green tea polyphenol epigallocatechin-3-gallate on high-fat diet-induced insulin resistance and endothelial dysfunction. Am. J. Physiol. Endocrinol. Metab. 305, 1444-1451.
4.Yao, Ting; Zhang, Chen-guang; Gong, Mong-tao; Zhang, Min; Wang, Lei; Ding, Wei (2016). Decorn-mediated inhibition of the migration of U8MG glioma cells involves activation of autophagy and suppression of TGF-B signaling. FEBS Open Bio. 10, 2211-5463.

SIBS Student: Hannah Newman
SIBS Mentor: David Schneider (dschneid@uab.edu)

There are three nuclear RNA polymerases expressed in Eukaryotes. RNA polymerase I is responsible for the synthesis of ribosomal RNA (rRNA) which is folded in the core of the ribosome. Ribosome synthesis is proportional to cell growth and proliferation. Due to this correlation, RNA polymerase I is emerging as a major cancer therapy and requires more characterization. Our lab characterizes RNA polymerase I function, using Saccharomyces cerevisiae as a model system. Currently, RNA polymerase I is isolated and purified from cells harvested during the log growth phase. This takes a large quantity of Saccharomyces cerevisiae to get a small amount of polymerase, which proves to not be cost effective or practical. The aim of this project is to create a purification strategy of RNA polymerase I using stationary phase cells to be able to recover larger quantities of active polymerase from smaller mediaquantities. This will be done using Fast Protein Liquid Chromatography (FPLC). We hypothesize that if RNA polymerase I can be isolated from the stationary phase, that it will be just as active from being purified from the log phase. Through this experiment, we were able to perform a purification thatresulted in partial purification, where protein was lost in one of the intermediate steps. We believe that with a different nickel column, protein would not have been lost. In the future, we hope to redo thisprocedure, while adding a Mono Q column after the final nickel column to ensure we only isolate our polymerase.

SIBS Student: Felicia Peoples
SIBS Mentor: John Shacka (shacka@uab.edu)
The Effects of HIV-Tat and Morphine on M17 Neuroblastoma Cells

Introduction: Parkinsonism has been reportedin HIV/AIDS, and alpha-synuclein (αsyn), involved in Parkinson’s diseasepathogenesis, ishigher in postmortem HIV patient brains. Many HIV/AIDS patients take chronic opiatesfor pain. Recent evidence indicates combined treatment with morphine and the HIV protein Tat exacerbatesneurotoxicity.Whether the opiate morphine+/-Tat regulates αsynmetabolismand autophagy lysosome pathway (which mediates αsyn clearance) markersin HIV/AIDS is unknown.

Objective: Determine if morphine+/-Tatregulates cell viability, levels of endogenous αsyn/ALP markers, or clearance of over-expressed αsyn.

Methods: M17 human neuroblastoma cellswereused that conditionally over-expresseshuman wild-type αsyn. Cells were treatedwith HIV-Tat(100nM)+/-morphine(0-500μM). Cell viability was measured viaMTT assay. Western blot analysis assessedlevels of endogenous αsynand LC3-II (autophagosome marker) and p62 (ALP substrate), andclearance of over-expressed αsyn.

Results: Morphine induced cell death at ≥100μM; Tat did not affect viability in the presence or absence of500nMmorphine. Western blot analyses suggested reductions inendogenousαsyn, LC3-II and p62 with500nM morphine ± 100nM Tat. 500nM morphine did not affect clearance of over-expressed αsyn.

Discussion: It appears that M17 cells are less sensitive toTat+morphine than cultured neurons,though previous studies have indicated that glial cells are important for mediating Tat +morphinetoxicity. Western blot results suggested that 500nM morphine+Tat induced ALP function. Ongoing studies are determining the effects of highermorphine concentrations on Tat-induced toxicity of M17 cells, and whether glial cells areimportant for eliciting these effects.

Key Words: HIV, Tat, Morphine, Alpha-synuclein, M17, Autophagy

SIBS Student: Samara Roman-Holba
SIBS Mentor: Sooryanarayana Varambally (soorya@uab.edu)
Selective targeting of histone methyltransferase EZH2 inhibits aggressive bladdercancer.

Histone methyltransferase EZH2 is overexpressed in multiple aggressive cancers including prostate, breast and bladder cancer. Increased expression of EZH2 in cancer leads to the down regulation of tumor suppressor genes such as E-cadherin as well as multiple microRNAs. Thus, therapeutic targeting of EZH2 may serve as an effective strategy in treating aggressive cancer. Treating bladder cancer cells with the EZH2 inhibitor GSK126 reduced bladder cancer cell proliferation, and in vivochickenembryo chorioallantoic membrane assayssuggested reduced tumor growth upon EZH2 inhibitor treatment. Gene expression profiling data using RNA from EZH2 inhibitor treated cells identified multiple re-activated target genes. We further validated one of the re-activated targets SQSTM1 by performing qPCR using RNA from EZH2 inhibitor treated cells and controls. In addition, we confirmed the re-activation by performing immunoblots using specific antibodies. Our investigations suggest that treating bladder cancer cells with an EZH2 inhibitor blocks cancer cell proliferation and invasion. Furthermore, we showed that inhibition of EZH2 results in the re-activation of multiple genes including autophagy regulator SQSTM1. In summary, our study suggests that targeting EZH2 may be an effective therapeutic strategy in treating aggressive bladder cancer.

SIBS Student: Pavitra Ravishankar
SIBS Mentor: Paul Goepfert

Streptococcus pneumoniae is a major cause of serious illness and death worldwide. Pneumococcal vaccines including Pneumovax 23 and Prevnar13 are indicated for young children and older adults with underlying medical conditions. Pneumovax 23 contains polysaccharide bacterial capsules from 23 different serotypes and is a “T cell independent” vaccine. This vaccine induces antibody only responses with an IgM isotype. Furthermore, there is no affinity maturation of these antibodies and development of B cells memory does not occur. In contrast, Prevnar 13 is a conjugate vaccine where polysaccharides from 13 different serotypes are covalently coupled with diptheria CRM197 as a carrier protein making it a T cell dependent vaccine. This vaccine is more immunogenic than Pneumovax 23 likely due to recruitment of CD4 T cell help. CD4 T cells provide direct (for e.g. CD40-CD40L interactions) and indirect help (secretion of cytokines) to B cells causing the latter to proliferate, mature (IgM to IgG isotype switching), elicit high affinity antibodies, and differentiate into long lived memory B cells that can rapidly response to secondary exposure of S. pneumoniae by producing antibodies. We hypothesize that higher immunogenicity of the Prevnar 13 vaccine will be observed among HIV seronegative individuals and that this is likely due to the preferential expansion of S. pneumoniae specific CD4 T follicular helper cells.

SIBS Student: Courtney Swain
SIBS Mentor: Yang, Yang (yangyang@uabmc.edu)
Osteoblast-derived Runx2 Regulates Bone MarrowCell Populations

Multiple myeloma (MM) is an incurable plasma cell malignancy that preferentially grows in the bone marrow (BM). MM frequently metastasizes from the primary bone site to new bone sites, resulting in a 5-year survival rate of only 43%. Within the BM, MM cells form complex interactions with resident BM cells (i.e. osteoblasts, osteoclasts, immune cells, etc.). MM cells remodel the extracellular matrix and secrete soluble molecules that favor further MM progression at both primary and secondary sites. Recent studies have demonstrated that in MM, osteoblast (OB) differentiation and bone formation are inhibited via the suppression of runt-related transcription factor (Runx2) in OBs. Importantly, we have shown that suppression of OB-Runx2 also occurs at distant sites prior to arrival of MM cellsin these sites. OBs, under the control of Runx2, also interact with other components of the BM microenvironment. The goal of this study was to investigate the effect of OB-Runx2 suppression on immune cells in the BM microenvironment.

To study the impact of OB-Runx2 suppression on BM microenvironment, we have developed an animal model in which Runx2 is specifically deleted in OBs (Runx2OB-/-). Tissues obtained from wildtype (Runx2 OB+/+) and homozygous (Runx2 OB-/-)C57Bl/KaLwRij micewere used in immunohistochemistry to observe different cell types within the BM microenvironment. Results demonstrated an increase in the population of cytotoxic T cells and a decrease in osteoblasts and macrophages in 5 week and 3 month-old Runx2 OB-/-mice, compared to Runx2 OB+/+control mice . Further studies will aim at determining immune regulatory cells, such asT reg cells, MDSCs,and soluble molecules in the BM microenvironment,that are altered byOB-Runx2 suppression.

SIBS Student: Roman Travis
SIBS Mentor: Ganesh Halade (halade@uab.edu)
Maresin-1 Directs the Resolution of Inflammation Post Myocardial Infarction

Introduction: Current inflammation inhibitory measures failed to improve healing post myocardial infarction (MI). Resolution of inflammation presents a unique avenue for clearing inflammation though the effects on heart failure pathology are unclear.

Hypothesis: Tested whether Maresin-1(MaR1)resolves post-MI inflammation thereby limitingpathological remodeling.

Methods: 8-12 week old male C57BL/6J mice underwent permanent coronary artery ligation surgery and were treated daily 3 hours post-MI with Mar1 (10ng/25g )until day1 (n=7) or day 5 (n=14). Mice with no treatment of MaR1 served as MI-controls,and mice with no-MI servedas d0 naïve controls. Post-MI neutrophils, macrophages and collagen density were measured using histology. Leftventricle (LV) function was measured using echocardiography Pro-resolving and pro-inflammatory immune cell markers were assessedusing real-time PCR followed by immunoblotting.

Results: MaR1 treated mice displayed increased neutrophil clearance at day 5 post-MI. MaR1 treated mice displayed no change in macrophage density, however, mRNA levels of pro-inflammatory markers i.e CD10, TNFα, IL-6,and IL-1βwere decreased at day 5post-MI (all p<0.05).Mice treated with MaR1displayedimproved cardiacoutput and ejection fraction. Post-MI MaR1 treated mice displayed increased levels of pro-resolving markers YM-1and Arg-1at day 1 post-MI with decreasedlevels of the lipoxygenase(Alox-12,-15 and-5)at day5 post-MIcompared with MI-control group. However, a post-MI fibrosis remained unaltered in MaR1 treated groupcompared with MI-control.

Conclusion: Maresin-1 .increases pro-resolving markers in post-MI heart failure pathophysiology thereby protecting heart function.

SIBS Student: John Treffalls
SIBS Mentor: Paul Gamlin (pgamlin@uab.edu)
Central projections of intrinsically photosensitive retinal ganglion cells in the Macaque monkey

The hypothalamic suprachiasmatic nucleus (SCN) is the master clock responsible for controlling circadian rhythms in mammals. These rhythmic cycles generated by the SCN are entrained to the environmental light/dark cycle via signals from intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin. Orexin is a neuropeptide that regulates arousal, wakefulness, and appetite. Orexingeric cells in the posterior hypothalamus receive input from the circadian system. It has been reported that orexin is present in melanopsin-expressing ipRGCs.However, most knowledge of these cells has been gained from studies on nocturnal rodents, whose visual systems are distantly related to those of humans and other diurnal primates. Injection of cholera toxin B (CtB), an anterograde axonal tracer, was used to visualize retinal projections in the brain, and anti-orexin antibodies were used to determine if these projections also contained orexin.One male macaque monkey, aged 20 years, receivedbilateral intra-vitreal injections of 100 μl of 0.5% CtB conjugated with either Alexa Fluor 488 (right eye) or biotin (left eye).After a survival time of 6 days, the animal was deeply anesthetized, perfused, and the brain and eyes were both removed. Immunohistochemical staining of revealed penetration of CtB into the retinalprojections of the brain, as well as the presence of orexin in the retina. This is the first study to observe orexin in the retina of nonhuman primates, supporting the hypothesis that orexinergic neurons in the SCN may be retinal projections of ipRGCs.

SIBS Student: Tuluca, Andrei
SIBS Mentor:Sabine Huke (huke2@uab.edu)
Assessing Pannexin 1 Channel Function in Mouse Cardiomyocytes

Pannexin1 (Panx1) is a ubiquitously expressed large pore conductance membrane channel that allows for the passage of small molecules like ATP. A role for Panx1 has been described during cell apoptosis, seizures, tumorigenesis, melanomas, migrainesand HIV-1. In addition, our lab has preliminary data suggesting that Panx1 facilitates Ca2+ dependent focalectopic arrhythmias. In order to assess Panx1 channel function in the heart it was necessary to isolate single mouse cardiomyocytes suitable for different assays. We chose to measure Panx1 channel function via patch clamp technique, which, requires stable membranes, and via ATP release from cell batches, which requires high cell yield. Cardiac tissues were separated and digested into individual cardiomyocytes with the use of a perfusion system that delivered 35-degree bubble free solution via a roller pump. Multiple collagenase “Cocktails” supplemented with protease to optimize for (1) highyield and (2) membrane stability were tested. We also explored if Panx1 channel internalization would hamper functional measurements by measuring the expression of the receptor responsible for internalization, P2X7, using qPCR. We identified cardiomyocyte isolation conditions that were suitable for patch clamping, receiving a 6 out of 10 score for patchability. Moreover, we identified conditions that yielded produced cardiomyocyte yields >30%?%. Taken together, even though time constraints prevented us to attempt Panx1 function measurements, cardiomyocyte isolation procedures are now in place for each assay. Our data also suggeststhat P2X7 is likely present on cardiomyocytes and the possibility of internalization during the isolation procedure needs to be requires future considerations.

SIBS Student: Yasmeen Abdo
SIBS Mentor: Akinyemi Ojesina (ojesina@uab.edu)

Background and Objective: Phenotypic characterization of mutations in cancer is important for understanding disease pathogenesis and developingtargeted therapies. The Ojesina laboratory recently identified recurrent hotspot mutations in the ZC3H11 Agene in cervical cancer (yet unpublished). This study aims to investigate the genomic and phenotypic consequences of ZC3H11 Amutations in cancer, and to test the hypothesis that the mutation from leucine to proline in position 801 (L801P) of ZC3H11A is associated with phenotypic features of cellular proliferation, motility,and invasiveness.

Methods: Using the Morpheus software, gene expression profiles were compared between 20ZC3H11A-mutant and 52 wildtype cervical tumors to identify upregulated and downregulated genes. The following phenotypic assays were performed on immortalized human embryonic kidney cell lines (HA1E) transduced with either the ZC3H11A wild type or L801P mutant: 1) the MTT assay to measure cell proliferation rates; 2) a wound assay to gaugecell motility;3) Chemotaxisand invasiveness assay using Matrigel.

Results: Gene expression profiling revealed that hemoglobin complex genes and polycomb target genes were upregulated while genes associated with the extracellular matrix were downregulated inZC3H11A-mutant tumors. Cell lines overexpressing the L801P mutant displayed higher proliferation rates than those harboring the wildtype gene. The mutant was also associated with increased cell motility but a lower invasiveness index.

Conclusions: These data are suggestive of oncogenic effects of somatic mutations in ZC3H11A. Ongoing and future experiments include reiteration of the already performed assays, soft agar colony formation assay and RNA sequencing of the cell lines.

SIBS Student: Katherine Ankenbauer
SIBS Mentor: Sooryanarayana Varambally (sorrya@uab.edu)
The role of PAICS in the tumorigenesis of pancreatic adenocarcinoma

Pancreatic cancer is an aggressive type of cancer and is the fourth most common cause of cancer-related deaths in the United States. Recent advances in high-throughput sequencing technologies helped identify numerous molecular alterations in pancreatic cancers including dysregulations in metabolic enzymes such as phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), an enzyme that participates in de novo purine nucleotide biosynthesis. Here, by using gene expression profiling and transcriptome sequencing datasets from pancreatic cancer, we show that PAICS is also upregulated in pancreatic adenocarcinomas. Furthermore, by silencing the PAICS using specific shRNA, we show a reduced cancer cell proliferation, invasion, and colony formation suggesting a critical role for PAICS in pancreatic cancer. Because it is enzyme involved in nucleotide biosynthesis, PAICS is amenable to small molecule inhibitors and can serve as a valid therapeutic target. Future studies will focus on inhibiting this enzyme with specific small molecules.

SIBS Student: Rachael Branscomb
SIBS Mentor: Ronadip Banerjee (ronadip@uab.edu)
Prolactin receptor signaling in the pancreatic β-cell: The role of STAT5

The lactogenic hormone, prolactin (PRL), has many critical functions during pregnancy including a role in the adaptive response of β-cellexpansion and glucose homeostasis in murine models. Previous data indicates targets of prolactin signaling in pancreatic β-cells include the genes Tph1, Hopx, and IVD. The prolactin signaling pathway through the prolactin receptor (PRLR) is mediated in part by transcription factor STAT5. The objective of this study is to determine if STAT5 is a direct mediator of gene transcription for these genes, further elucidating the signaling pathway of the PRLR and its gene targets. We used both a mouse insulinoma cell line (MIN6) and isolated mouse islets, treated with and without PRL. The localization of STAT5 binding was detected with the chromatin immunoprecipitation (ChIP) assay, a method that allows for the direct detection of protein-DNA interaction, using a rabbit α-Stat5a/b antibody to immunoprecipitate. With overnight PRL treatments, Tph1 expression increased 50+ fold in islets and 6+ fold in MIN6. When compared to control IgG antibody, PRL treatment resulted in a 3-fold enrichment of the STAT5 response element, upstream of the Tph1 gene in MIN6 cells. Enrichment at the STAT5 response element suggests that STAT5 is a mediator for Tph1 expression. Further method refinement is needed for successful islet ChIP. Validation of STAT5 binding for Hopx and IVD is ongoing.

SIBS Student: Elizabeth Dyer
SIBS Mentor: Craig Maynard (clmaynard@uab.edu)
Understanding the Role and Relationship of IL10 and ICOSL in Inflammatory BowelDiseases

Inflammatory Bowel Diseases (IBD) are multifactorial diseases involving chronic inflammation of the digestive tract. Genome-wide association studies (GWAS) have identified almost 200 genes that can impact susceptibility to IBD. We examined the possible collaboration between 2 IBD susceptibility genes, the inducible co-stimulator ligand (ICOSL) and interleukin-10 (IL10), in preventing intestinal inflammation. Previous studies have shown that ICOSL co-stimulation promotes elevated expression of IL10. However,our lab has found that ICOSL signaling is not required for induction of Il10by intestinal CD4 T cells. In fact, genetic deletion of ICOS, the receptor for ICOSL, results in increased IL-10 expression throughout life. In this study,we analyzed whether co-inactivation of the ICOSL and IL-10 pathways in mice results in accelerated development of intestinal inflammation. Mice that are either wild type or deficient for ICOSL were injected, or not, with an antibody that prevents binding of IL-10 to the IL-10 receptor (IL-10R). To monitor the development and progression of inflammation, the mice were weighed and the concentration of lipocalin-2 (Lcn-2) in feces was monitored every 5 days. Immunological analysis of gut CD4 T cells was performed at the end of the study. ICOSL-deficient mice injected with IL-10R blocking antibody had elevated levels of fecal Lcn-2 and demonstrated enhanced weight loss relative to the other groups. Also, lamina propria CD4 T cells produced elevated levels of IL-17 and IFNγ. Collectively, our data suggest that deficiencies in both ICOSL and IL-10 predispose to rapid onset of intestinal inflammation in mice.

SIBS Student: Gunnar Eastep
SIBS Mentor: Jamil Saad (saad@uab.edu)

Retroviral replication depends upon targeting of the viral structural protein Gag at the inner leaflet of the plasma membrane (PM), leading to the assembly and budding of immature virus particles. The N-terminal matrix (MA) domain of Gag interacts with thePM through a combination of hydrophobic, electrostatic and lipid-specific signals which vary in their contributions among retroviruses. It is well established that a highly basic region (HBR) is a conserved MA feature and is essential for Gag binding to PM through non-specific electrostatic interactions. Additionally, it has been demonstrated that PM targeting of HIV Gag depends on specific interactions of the MA domain with PI(4,5)P2present in the inner leaflet of PM. While it has been shown that the binding of avian sarcoma virus (ASV) Gag to lipid bilayers is enhanced by PI(4,5)P2, the role of this lipid in ASV Gag localization to the PM remains controversial. Using nuclear magnetic resonance and a recently developed liposome binding assay we seek to understand the strength, specificity and structural aspects of ASV MA interaction with PI(4,5)P2, including possible roles of other PM components. Our results are consistent withan electrostatic model of binding where increasing total negative charge of phospholipids increases the strength of the interaction with RSV MA. However, still more work is required to find a way of liposome preparation that would assuregood reproducibilityof the binding experiments.

SIBS Student: Kaelee Hale
SIBS Mentor: Mentor: A. Brent Carter (brarter1@uab.edu)

Background

Pulmonary fibrosis is a disease characterized by irreversible remodeling of the lung tissue. Macrophagesare critical in the progression of pulmonary fibrosis. A characteristic feature in macrophages in fibrosis is increased mitochondrial (mt) ROS.The Rho GTPase Rac1 induces macrophage mtROS and is required for the development of pulmonary fibrosis. Although mtdysfunction is associated with apoptosis in many cell types, macrophages from human subjects with fibrosis are resistant to apoptosis.

Hypothesis

Rac1 modulates mtdynamics and metabolic reprogramming to fatty acid oxidation (FAO) to mediateapoptosis resistance in lungmacrophages.

Methods

Expression ofconstitutive active Rac1 (Rac1CA)was utilized. Mitophagy was analyzed using immunoblot analysis and TEM. Mitochondrial biogenesis was determined by confocal microscopy and RT PCR. A Seahorse Bioanalyzer was used to measure FAO. WT mice and Rac1-/-Lyz2-cremicewere used for in vivo studies. Apoptosis was determined by caspase-3 activity and TUNEL.

Results

Macrophages treated with chrysotile or expressing Rac1CA displayed increased expression of mitophagy-associated proteins including p62, PINK1, and LC3 II. WT mice exposed to bleomycin had increased mitophagy. Chrysotile increased expression of TFAM. FAO was increased byRac1. Macrophages from WT mice were resistant to apoptosis, whereas macrophages from Rac1-/-Lyz2-cremice were apoptotic.

Conclusions

Mitophagy and mitochondrial biogenesis are greaterwith over-expression of Rac1CA. Over-expression of Rac1CApromotedFAOin macrophages. Lung macrophages from WT mice have increased mitophagy and are resistant to apoptosis, while macrophages from mice harboring a deletion of Rac1 lack mitophagy and have increased apoptosis.

SIBS Student: Joline Hartheimer
SIBS Mentor: John Hartman (jhartman@uab.edu)
Cellular Mechanisms Underlying Gene-Nutrient Interactions Affecting Longevity

Introduction: Aging is associated with declining function and increased risk of disease. Dietary interventions are known to modulate lifespan and health span,yet the cellular mechanism sof nutritional influences on aging are not well understood. Saccharomyces cerevisiae can be used to measure aging by calculating Chronological Life Span (CLS), the amount of time that a non-dividing yeast cell survives. CLS can be measured using Quantitative High Throughput Cell Array Phenotyping(Q-HTCP) which uses robotics and computational analysis. Of great interest to aging in eukaryotes is the fraction of quiescent cells in stationary phaseyeast culture that have exited the cell cycle due to nutritional stress, but are still capable of reentering upon rich nutritional conditions.

Objective: To identify gene-nutrient interactions affecting S. cerevisiaeon chronological life span and better characterize their cellular mechanisms.

Methods: CLS was measured by Q-HTCP to identify aging phenotypes resulting from different auxotrophic alleles of S. cerevisiaeon human-like media with different concentrations of carbon (dextrose) and nitrogen (ammonium sulfate). Mitochondrial activity and cell cycle distribution (G1/G0 arrest) were assessed with flow cytometry.

Results:Dextrose restriction and methionine auxotrophy protected against aging over 28 days. Ammonium sulfate’s effects depended on auxotrophy and dextrose concentration. In two auxotrophic strains, mitochondrial activity decreased over 6 days. Prototrophs hada more stable quiescent (G0) state than auxotrophs over 7-10 days.

Conclusion: Complex gene-nutrient interactions exist in yeast and their mechanisms will be further analyzed with multiwell and multiplex flow cytometric assays with respect to apoptosis, bud scars, and lysosomal acidity.

SIBS Student: Danielle Kem
SIBS Mentor: Andrew West (abwest@uab.edu)

Background

Thealpha-synuclein(α-syn)fibril model of Parkinson’s disease(PD)is used to induce PD like pathology in ratsin ordertostudy PD and test drug candidates.In thismodel, sonicated α-syn fibrils are injected into the substantia nigrapars compacta (SNpc) region of the brain. These fibrils are taken up by neurons where they corruptthe endogenous α-syn causing them to aggregate into inclusions called Lewy Bodies(LB). Inclusion formation is accompanied by progressive loss of dopaminergic neurons, and SNpc denervation of thestriatum.

Objective

We are determining if 23nm α-synuclein fibril fragments are more effective at seeding PD pathology than 49nm fragments. The optimized model will be used to test PD drugs.

Methods

40μm rat brainssections containing the SNpc and striatum were collected. These slices were then stained and used to quantifythe loss of dopaminergic neurons, quantify LB aggregation in the SNpc and striatum, and quantify denervation of the striatum bydopaminergic neuronsresiding in the SNpc.

Results

There was a remarkable 50 fold increase in the number of inclusions in the striatum of the 23nm injected ratsover the striatum of rats injected with 49nm PFFs, while the number of inclusions in the SNpc and the TH+ fiber density of the striatum remained the same in both groups of rats. Both showed 40% loss of dopaminergic neurons in the SNpc but variability was halved in the 23nm cohort.

Conclusions

The 23nm α-synuclein fibrils were more effective in seeding PD pathology than the 49nm fibrils.

SIBS Student: Sheila Mallenahalli
SIBS Mentor: Colin Reily (creily@uab.edu)

Introduction
IgA Nephropathy (IgAN) is the leading cause of primaryglomerulonephritis. Elevated levels of Galactose deficient IgA1 (Gd-IgA1) antibody react with IgG autoantibodies, formingcirculating immune complexes (CIC) that deposit in the kidney, causing decreased kidney function. IgAN patients often present with synpharyngitic hematuria, suggesting an inflammatory component. In vitro, IL-6 induces Gd-IgA1 production in immortalized B cells from IgAN patients, but not controls. This study teststhe effectsof othercytokines, primarilyIL-4, IL-21 and CD40L,on Gd-IgA1 productionand attemptsto identify downstream signaling processes responsible for increased autoantigen secretionfrom both IgAN patients and controls. Pinpointing abnormal inflammatory responses may lead to better treatment regimens,as IgAN currentlyhas nodiseasespecific therapy.

Methods
EBV immortalized Bcells from IgAN patients and healthy controls were exposed to an initial cytokine stimulus (IL-4 at 100ng/mL, 50ng/mL, 25ng/mL; and IL-21 and CD40L at 50ng/mL, 25ng/mL, 12.5ng/mL) and incubated for 4 days. Cells were pelleted and lysed for western blotting,and media was harvested for IgA analysis. IgA production was assessed with an in-house IgA ELISA. Gd-IgA1 was assessed througha lectin based ELISA.

Results
Exposure to IL-4, IL-21 and CD40L showed no significant change in IgA1production, but a trend towards decreased Gd-IgA1productionin controlsand increased Gd-IgA1productionin IgAN samples.

Conclusions
Unlike IL-6, IL-4 IL-21 and CD40L had no effect on IgA1 production.orHowever, exposure from these cytokines showed a trend toward decreased galactosylationin IgAN cells. This increased Gd-IgA1 production may lend insight to disease specific galactosylationpathways.

SIBS Student: Nidhi Manu
SIBS Mentor: David A. Brown (dbrownpt@uab.edu)

Purpose:The goal of my project wasto analyze data from a completed training study thatusedtwo body-weight-supported treadmill-training protocols (i.e., Hands-Free (HF) and Skills-Based (SB)). Both useda novel robotic-treadmill interface(KineAssist), to improve walking outcomes for stroke survivors.We hypothesizedthat 1) the HF group would spend more timeat the desired heart rate (HR) intensity, 2) both groups would need decreasedbody-weight-support(BWS)levels over training, and 3) there would be an increase in distancetraveled and steps taken per session, but to a greater extent in the HF group.

Methods:Using the KineAssistto provide BWSand a safe walking environment, we randomized 39poststrokeparticipants into two training groups. The HFgroup trained without handrails, while the SBgroup trained in nineessential walking skills also withouthandrails. Both groups trained for 30 minutes at 60-80% of their target HRreserve/session, threetimes/week for sixweeks. During each session, we measured participants’ HRevery minute, andrate of perceived exertion (RPE) every twominutes. We recorded BWS levels, total distance traveled, and number of steps taken per session.

Results:Our results showed that neither group adhered well to the target HRrange, but that both perceived their exertion to be at least somewhat hard for mostof training. Participants in both groups needed less BWSover time,and walked farther and with more steps over the course of training. The increases in distance traveled and steps taken were similar for both groups.

Conclusions:Both groups demonstrated poor HR adherence, but reported high RPE levels. This disconnect between actual and perceived intensity might be due to the challenge of walking with a weaker limb, but this finding requires further investigation. Thesimilar between-group improvementsin distance and stepsmay be one reason for our lack of observed group differences in overground walking outcomes.

SIBS Student: Joshua Nougaisse
SIBS Mentor: Stephen Aller (sgaller@uab.edu)
Crystallization of High Affinity Copper Uptake Protein (hCTR1) and Structural Analysis Using X-ray Crystallography

Introduction/Background:CTR1 functions as a high affinity copper uptake protein and is the only protein of its class in the human genome. Although there are few studies on CTR1, current evidence supports the idea that the protein acts as a transporter, but is structurally likea channel. This structural paradox alone poses several questions about the nature of the regulation and uptake of copper in humans.

Objective:The goal of this project is to crystallize a purified CTR1 protein and find out the structure and binding mechanisms of CTR1 at high resolution. Based on processed data, steps can be taken to find a mechanism of this protein.

Methods:Yeast expressing the gene for CTR1 were prepared and the protein was extracted and purified. The purified protein was then grown into acrystallized state under various conditions. Finally x-ray crystallography was used to collect data on the refraction patterns of the protein.

Results: X-ray crystallography data reveals an untwinned crystal of CTR1 at high resolution.

Discussion: Currently, there is insufficient data to determine a complete structure of CTR1. Although, the methods in this project resolved the twinning problem that appeared in earlier attempts of this crystallization process. Growing untwinned crystals will eventually allow a complete structure for this protein to be solved. These results will play a significant role in creating novel anti-cancer drugs as well as identifying the exact process of copper transport in the body.

SIBS Student: Christopher Nutter
SIBS Mentor: Jianyi Jay Zhang.(jayzhang@uab.edu)

Ischemic heart disease is one of the leading causes of death worldwide. In the last several decades, induced pluripotent stem cell-derived cardiomyocytes have been shown to exhibit a potential way of treating ischemic heart disease without the need of a complete heart transplantation. One major obstacle for these IPS-CMsis their immature phenotype, with previous work showing that transplantation of these cells induces cardiac arrhythmia. Therefore, a need for maturation before transplantation is requiredfor tissue regeneration to become successful. Several studies have implemented “natural stressors” to help emulate the environment that an in-vivo cardiomyocyte is subjected to. This study’s aim is to induce IPS-CM maturation through electrical stimulation while concurrently applying genetic modifications to enhance cell proliferation. Overexpression of cyclin D2, a member of the D-type cyclins involved in cell cycle regulations, has been shown to increasecell proliferation. We hypothesizethat inducing maturation through electrical stimulation coupled withthe overexpression of cyclin D2willincrease maturationand cell cycleof cardiomyocytes.After exposing the cardiomyocytes toan electrical stimulation mode of 5 Volts, 5 millisecond pulses, and 2 Hertz for a period of 7 days, our results indicate partial maturationof cardiomyocytes via an immunohistochemistryassay. Also, we found that an increasematuration didn’t significantly reducecell cycle of the cyclin D2 strains compared to the wild type control cells.In the future, we hope these results will give us a better understanding of the conditions required for a successfully higher yield of IPS-CM transplantations in the future.

SIBS Student: Kush Patel
SIBS Mentor: Mark Bevensee (bevensee@uab.edu)

The Na/bicarbonate cotransporter NBCe1 regulates pHandfacilitatessolute reabsorption/secretionin tissues, including bicarbonate reabsorption inthe kidney. Currently, there is little structural information about NBCe1. We have generated homology models of NBCe1thatrevealhow specific transmembrane domains, including TMD12,interactto translocateions.The purpose of this projectisto validate TMD12’s role in ion translocation.

Cysteine-scanning mutagenesis with sulfhydryl reagents(i.e.,MTSEA)wasused to determine if specific residues of TMD12 are involved in ion translocation. Individual residues werereplaced by cysteine, and the mutant proteins wereexpressed in Xenopus oocytes and studied functionally using voltage-clamping techniques to measure NBCe1 current.Experiments wereperformed to determinethe activity of mutant proteinsand their sensitivityto MTSEA.MTSEA inhibition of NBCe1 function implicatesthose mutated residues lining the translocation path.

Results from experiments designed to examine the five most cytosolic residues of TMD12 are consistent with TMD12 contributing to the ion translocation path and oriented in our modeled position. M868Cfacing the path gave a functional and modestly sensitive NBCe1 to MTSEA; A871Cangled away from the path generated an NBCe1 with inconsistent sensitivity to MTSEA. G869Cand S872facing the path produced nonfunctional constructs, implicating thoseresidues being critical for ion translocation.

These data help identify specific residues and the orientation of TMD12 that contributes to the ion translocation pathwayof NBCe1. Overall, this study will help advance our structure-function understanding of this protein, as well as the development of specific transport inhibitors/stimulatorsto treat disease states.

SIBS Student: Rachel Sutton
SIBS Mentor: Michael Gray (mjgray@uab.edu)

There has been a long-standing observation that fermented foods provide health benefits in the human gut. The Aryl Hydrocarbon Receptor (AHR) found in the human gut has also been found to promote a healthier gut by reducing inflammation and the risk of cancer. The AHR is activated by a variety of molecules, which are termed AHR agonists. Precursors of AHR agonists found in vegetables, which include glucosinolates, are thought to be converted into AHR agonists by the action of enzymes or bacteria, either in foods or in the microbiome.We hypothesize that the lactic acid bacteria (LAB) in fermented foods might convert glucosinolates to AHR agonists which may promote a healthier gut. In collaboration with Dr. Greg Kennedy, UAB division of Gastrointestinal Surgery, we tested levels of AHR agonists and bacterial populations over the course of a natural sauerkraut fermentation. We prepared sauerkraut and measured AHR agonist activity at different time points using a dioxin response element luciferase assay, which allowed bioluminescence to be recorded as an indicator of AHR activity. Results from the assay showed very low levels of AHR activity around 1 (fLUC/rLUC). We will run the assay again with samples that have a neutral pH and a higher concentration to test whether our hypothesis is correct. If the assay is successful, and AHR activity is increased throughout the fermentation process, it may lead to further studies regarding AHR activation and fermented foods.

SIBS Student: Samantha Yates
SIBS Mentor: Daniel Gorelick (danielg@uab.edu)
The G protein-coupled Estrogen Receptor is Required for Normal Swimming Performance in Zebrafish

Estrogens regulatethe development and function of the cardiovascular system via activation of nuclear estrogen receptors alpha and betaand the G protein-coupled estrogen receptor (GPER). Zebrafish embryos with a deletionmutation inthegpergene have reduced heart rate relative to wildtype. Reduced heart rate in embryos mayinhibit proper cardiovascular development, leading to reduced cardiac outputin adults.The goal of my research is to determine the effect of the GPER mutation on cardiac output in adult zebrafish.Cardiac output is difficult to measure in free-swimming zebrafish. However, previous research demonstrated that a measure of swimming performance, critical swimming speed (Ucrit),which can be readily measured, is directly proportional to cardiac output.We therefore used theswim tunnel assay to measure the Ucritof wildtype and maternal zygotic gper mutant fish(MZgper-/-) at 12 and 18 months of age.We found that MZgper-/-males had significantly lower Ucritthan wildtype malesat both ages tested. 18 month oldMZgper-/-females had significantly lower Ucritthan same-aged wildtype zebrafish, but we did not find a significant difference between the Ucritof 12-month female and MZgper-/-mutantand wildtypefish. These results indicate that gperis required for normal cardiac output in agedzebrafish. Additionally, the cardiac phenotype presents earlier in male zebrafish, suggesting a more prevalent role for gperin male cardiac function. Future studies will performhistological analysis on MZgper-/-zebrafish hearts to detect anatomical defects that may contributeto reduced cardiac output, such as reduced valve width.

SIBS Student: Tanya Zubov
SIBS Mentor: Shannon Bailey (sbailey@uab.edu)

Alcohol damages the multiple organs in the body. The circadian clock is important for maintaining diurnal rhythms in metabolism and health, whereas, disrupted clocks cause disease. The effect time-restricted alcohol feeding has on liver and brain is not known. Our goal is to examine the impact time-restricted alcohol feeding has on clock genes in liver and hippocampus. We hypothesize that there are time-of-day differences in how alcohol alters the clock and metabolism.

Mice were housed under a 12:12 h L:Dcycle and were fed control or alcohol diets. Mice were maintained under two time-restricted feeding models: Dark-phase time-restricted feeding (ZT 12 –24) or light-phase time-restricted feeding (ZT 0 –12). At the end of the feeding protocol, liver and hippocampus were collected and mRNA levels of clock and metabolic genes were measured.

We observed alcohol and time-of-day differences in expression of several genes. Alcohol increases and decreases Bmal1 and Per2 levels in liver and hippocampus, respectively. Pgc1a levels in liver were higher in night-fed mice vs. day-fed mice, with no difference in hippocampus. Sirt1 levels were similar in all groups, whereas Prkaa1 levels were increased and decreased in livers of night and day-fed alcohol mice, respectively. Alcohol decreased Prkaa1 in the hippocampus.

The results from this study are the first to report the effect of time-restricted alcohol consumption on clock gene expression. These studies demonstrate that there are time-of-day differences in how alcohol affects the clock. Our findings will advance understanding of the mechanisms responsible for alcohol-induced tissue injury.

Rachel Dobrozsi
SIBS Mentor: Jillian Richter (jillianrichter@uabmc.edu)
The Effect of Resuscitation Fluids on the Release of Heparanase & Its Relation to Syndecan Shedding

Introduction: ​Trauma is the leading cause of death for those under 45 years old in the UnitedStates. In some cases of trauma, hemorrhagic shock occurs, causing a long chain of pathologicalreactions that ultimately produce an inflammatory response within the human body. Duringinflammation, the protective barrier covering the endothelium (the glycocalyx) plays animportant role in vascular permeability. It is a regulator of leukocyte and platelet interaction withadhesion cell molecules on the endothelial surface, thus influencing local inflammatorysignaling. During trauma, platelets and leukocytes adhere to the endothelium, inducing a furtherrelease of cytokines, proteases, and the enzyme heparanase (HPSE). The enzyme Heparanase hasemerged as a major regulator in the inflammatory process by degrading heparan sulfate, causingsyndecan shedding and damage to the glycocalyx, ultimately exposing the endothelium. HPSE isbiologically important and, in recent years, it is believed that the release of HPSE from plateletsmight be activated by different resuscitation fluids used in caring for trauma patients. LactatedRingers (LR) which are believed to possibly damage the glycocalyx and fresh frozen plasma(FFP), which has been shown to preserve the glycocalyx, will be added to mouse platelets todetermine whether they cause platelets to release HPSE.

Objective: ​This study will examine HPSE released from platelets when exposed to multipleresuscitation fluids commonly used in an emergency setting. The amount of HPSE released fromthese platelets will determine if this enzyme is causing syndecan to shed and ultimatelydamaging the Glycocalyx. The results of this study can be used to better understand HPSErelease of platelets and to determine whether or not different resuscitation fluids can preserve theglycocalyx, improving trauma outcomes.

Methods: ​Whole blood was extracted from mice and their platelets were isolated, then treatedwith either LR or FFP for 0.5, 1 or 4 hours. Thrombin was the positive control and PBS was thenegative control. After treatment, cellular and extracellular HPSE levels were measured withWestern blot and immunoblotting, respectively. Platelet media was added to mouse endothelialcells and either treated, or not treated with OGT-2115, a HPSE inhibitor. The loss of syndecanfrom the cells was then measured to determine glycocalyx damage from HPSE.

Results (expected): ​It is expected that the LR will cause a greater release in HPSE from plateletsthan the FFP. The release of HPSE on glycocalyx damage will be confirmed when the inhibitionof HPSE with OGT-2115 shows no syndecan shedding, while no inhibition of HPSE confirmssyndecan shedding.

Conclusion:​ FFP did not cause a release of HPSE, confirming that the use of FFP improves theultimate outcomes of trauma patients by not damaging the Glycocalyx and exposing theendothelium. These results can be used in a hospital setting to potentially inhibit HPSE duringresuscitation which can then limit glycocalyx damage and prevent secondary complicationsrelated to organ dysfunction after trauma.

Zoe Fokakis
SIBS Mentor: Farah Lubin (flubin@uab.edu)
Classical Conditioning of Electroconvulsive Seizures as a Model of Non-Epileptic Seizures

Epilepsy resultsfrom abnormal circuit activity in the brain; however, in 75% of patients with epilepsy, the cause is unknown.This has resulted inas many as 20-30% of patients being misdiagnosed with the neurological disorder. One hypothesis to explain these high rates of misdiagnosis is that some patients are sufferers of non-epileptic seizures (NES). One major form of NES is called ‘psychogenic seizures’ that are caused by mental or emotional processes, rather than abnormal electrical activity. The reason for this emotional association is unknown due to a lack of research.The goal of this study is to develop a ratmodel for NESthrough classical conditioning. A cohort of 20 Fischer-344 rats will be divided into four groups: 1) home cage (HC) rats that will be handled and habituated to transport, 2) an experimental group (EX) that will be presented with a tone (CS) before experiencing the electroconvulsive seizures (US), 3) a conditioning control (CC) group that will be experiencethe CS but not the US, and 4) an unconditioned control (UC) group that will experience the US but not the CS. Each group will experience classical conditioning of seizures for five days. Forty-eight hours later, rats will experience the CS alone for five consecutive days. One hour after exposure of rats to the CS on Day 5, rats will be euthanized and the hippocampus isolated for evaluation of posttranslational histone modifications.

We predict that EX group rats will experience the full spectrum of the Racine scale when presented with only the CS. No other group of rats should seize during the testing period. EX rats will demonstrate no extinction after five days of testing with the CS.Results from this study will determine whether convulsive seizures can be learned through classical conditioning.

Jonathan Hanan
SIBS Mentor: David Schneider (dschneid@uab.edu); Catie Scull (cescull@uab.edu)
Construction of Pol I Active Center Mutant

Eukaryotic cells express three unique nuclear RNA polymerases that have distinct gene targets, transcription machinery, and core enzyme composition. RNA Polymerase I (Pol I) functions to synthesize ribosomal RNAs, the molecules that make up the majority ofribosome structure and carry out ribosomal functions.The active center of Pol I contains a Pol I-specific core subunit A12.2, which is essential for Pol I innate nucleolytic activity and profoundly affects nucleotide incorporation and elongation complex stability. The C-terminal domain (CTD) of A12.2 is necessary for its nucleolytic activity. Saccharomyces cerevisiae (budding yeast) mutant with deletions of A12.2 C-terminal domain (rpa12∆CTD) will be constructed to determine the effect of A12.2 CTD on Pol I enzymatic activity, specifically the influence of A12.2 CTD on polymerase transcription elongation and elongation complex stability.

Shelby Harris
SIBS Mentor: A Brent Carter (acarter@uabmc.edu)

Introduction: Idiopathic pulmonary fibrosis (IPF) is a diseasecharacterized by the formation and the build-up of scar tissue in the lungs. The lungs become stiff andlung function and gas exchangeare reduced. There is no known cause of IPF,and current treatments for the disease are not effective in stopping the progression of IPF.
Objectives: This project investigatesa pathway bywhichRac1, a small GTPase,induces mitochondrial reactive oxygen species (ROS)that results in mitochondrial biogenesis and alternative activation of lung macrophages to a pro-fibrotic state in pulmonary fibrosis.
Methods: For these studies, THP-1 and MH-S cells were transfected with four different plasmids: Empty (control), Rac1WT(wild type),Rac1CA(constitutively active), or Rac1DN(dominant negative). Mitochondrial samples isolated from transfected cells were usedfor immunoblot analysis to confirm overexpression of Rac1 and the localization of Rac1to the mitochondria.The generation of hydrogen peroxide in the mitochondriawas determined by pHPA assays.RNA was also isolated from cells for real-time PCR to measure the effect of Rac1 on gene expression. MitoTrackergreen was used to stain mitochondria in transfected cells.
Results: Immunoblot analysis showed that Rac1 was over expressed with transfection of Rac1WT, and it localized to the mitochondria. RT-PCRrevealed that fibrotic genes, such asYm-1 andTGF-β1, were increased in cells expressing Rac1WT compared to the empty control. The pHPA assay demonstrated that over expression of Rac1WT in cells generated significantly more H2O2 than the cells expressing Rac1DNor empty. Silencing Rac1 with siRNA decreased fluorescence of MitoTrackergreen, while overexpression of Rac1CA increased TFAM in mitochondria. In contrast,cells expressing Rac1DNhas a marked decrease inTFAM.
Conclusion: Mitochondrial ROS in macrophages has a critical role in the pathogenesis of pulmonary fibrosis. We found that the mitochondrial localization of Rac1 increases mitochondrial ROS, which mediated mitochondria biogenesis and increased expression of pro-fibrotic factors in macrophages. These data suggest that Rac1-mediated mtROS plays a vital role development of pulmonary fibrosis

Veena Krishan
SIBS Mentor: Ganesh Halade (ganeshhalade@uab.edu)

Introduction: Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) are commonly used to controlpain,inflammation, and limit cardinal signs ofinjury in humans. However, use of NSAIDs increases the of risk heart attack (myocardial infarction; MI) and subsequent risk ofrenal failure.
Objectives:The molecular and cellular mechanism of actionfor this adverse effect, particularly along the cardiorenal axis, is incomplete.
Methods: To define the mechanism, carprofen (CAP; NSAID in animals) was administered at 5 mg/kg body weight to C57BL/6 mice for two weeks. Some of the mice were then subjected to MIsurgery, whilemaintaining naive-controls. After occlusion of left anterior descending coronary artery, kidney histology and injury markers such as serum creatinine and neutrophil gelatinase associated lipocalin (NGAL) were examined. Biomarkers of inflammation such as TNF-α, IL-1β and COX-2 expression levels were also measured, and left ventricle histology performed to validate MI surgery.
Results: CAP treatment increased plasma serum creatininelevels. The myocardium showed structural disorganization of the fibers post-MI. Kidney NGAL mRNA and protein expression were increased post-MI. Post CAP treatment, TNF-α, IL-1β, and COX-2 expression levels increased prior to MI but failed to increase post-MI indicative of impaired initiation of the normal inflammatory response. There was also evidence that renal damage post-CAP treatment and post-MI was much greater, as plasma creatinine and NGAL levels were elevated with severe structural damage of the glomerulus.
Conclusion: Therefore, CAP treatment tampers with the acute inflammatory response and increases cardiorenal syndrome post-MI, amplifying the damage done by an MI.

Christopher Lightfoot
SIBS Mentor: Alexander Szalai (aszalai@uabmc.edu)

Introduction: Acute kidney injury (AKI) is the sudden loss of renal function due to ischemic damage to renal tubular epithelial cells (RTECs). Elevation of bloodC-reactive protein (CRP), a biomarker of inflammation, is correlated to worse prognosis in all stages of AKI. Previous studies in the Szalai laboratory using human CRP transgenic mice (CRPtg) showed that CRP exacerbates renal ischemia reperfusion injury, apre-clinical model of AKI. However, it has not been shown whether CRP has a direct impact on RTECs. To investigate the role of CRP on RTEC health, we studied theiradherens junction protein E-cadherin.

Objective: To identify the causal mechanism supporting CRP-mediated exacerbation of AKI and to assess CRP’s impact on RTEC health.

Methods: Primary mouse RTECs were isolated, cultured, and exposed to doses of CRP.E-cadherin integrity was examined using confocal immunofluorescent microscopy and expression was quantified by a western blot.

Results: Primary mouse RTECs showed robust E-cadherin staining at the cell membrane, indicating intact cell-cell contact. When treated with 50 μg/ml CRP for 24 hr, RTECs had no overt differences in expression patternhowever preliminary data indicatea 1.92-fold increase in E-cadherinexpression.

Conclusion: Our datashowed that when exposed to CRP, RTECs do not alter their E-cadherin orientation yet the overall expressionof E-cadherin increased. CRP also increases RTEC entrance into S phase (data not shown). Our current hypothesis is that CRP increases RTEC replication resulting in greater E-cadherin expression.Future research will evaluate how CRP,with other immune mediators,effectsRTECs during AKI.

Beverly Lincoln
SIBS Mentor: John Hartman (jhartman@uab.edu)
Effects of Auxotrophy and Media Composition on Yeast Lifespan and Production of Aging Factors

INTRODUCTION: Budding Yeast (Saccharomyces Cerevisiae) is commonly used as a model organism for aging and displays variation in longevity with respect to nutrition and ability to produce amino acids.

OBJECTIVES: The primary objective of this study was to examine if lifespan differed under varying carbon and nitrogen source allotment, and if yeast metabolites could affect fly longevity.

METHODS: Two yeast Q-HTCP CLS experiments were set up. The first consisted simply of the 6-day old CLS flask cultures (harvested for fly food), transferred to 384-culture cell arrays (using only the FY4 strain). The 2ndconsisted of fresh CLS cultures with the four expired HL media organized by quadrants on plates with a base of fresh media, where each quadrant contained different expired media (0.4% glucose without Ammonium Sulfate, 4.0% glucose-No Ammonium Sulfate, 0.4% glucose-Ammonium Sulfate, 4.0% glucose-Ammonium Sulfate) added, also including a fifth aging array with water added instead of fresh media. The four fresh media conditions plus water acted as a base on the plates, with the 4 expired medias added on top in a quadrant fashion. Two plates were observed for each base media, resulting in 10 plates total. With frequency at least once per week, CLS was assayed using Q-HTCP to collect growth curves generated after transferring aging cell cultures to fresh HLD (2% dextrose without ammonium sulfate) agar media. Throughout its entirety, the 2ndconditioned media experiment was hand-assayed using a manual pin tool. The “L” parameter from fitting growth curve data to the logistic function, (G(t)= K/(1 + e-r(t-l)), was used to estimate CLS.

RESULTS: Fly survivorship gave insufficient results to assume an affect of yeast metaboliteson fly lifespan.In both CLS experiments, cultures containing Ammonium Sulfate decreased yeast longevity.Results from the Conditioned Media CLS experiment suggested conditioned media had no effect on fly lifespan.

CONCLUSION: Ammonium Sulfate does not support CLS. The affect media conditions have on yeast lifespan should be further studied and for longer amounts of time to get any significant findings. Although no significant association was shown between yeast metabolites and fly lifespan, this study acts as a good model for future yeast-fly studies.

Benjamin Nelson
SIBS Mentor: Michael Gray (mjgray@uab.edu)

Reactive oxygen species such as hydrogen peroxide (H2O2) are produced as part of the innate immune system’s response to bacterial invasion. Hallmarks of intestinal inflammation, these highly reactive oxidants also attack the body’s own intestinal tissues and can lead to inflammatory bowel diseases such as Crohn’s disease, ulcerative colitis, and, when chronic, colorectal cancer. Lactobacillus reuteriis a Gram-positive probiotic bacterium known to ameliorate these potentially dangerous conditions. However, knowledge of how L. reuterisenses and responds to inflammatory oxidants is limited. Our lab’s previous research using whole genome transcriptomic analysis has determined multiple genes that are differentially regulated in L. reuteri’s response to H2O2 under microaerobic conditions. Based on the transcriptomic analysis, a number of null mutants of potentially redox responsive genes have been developed using oligo-directed recombineering. This study seeks to functionally characterize some of these redox responsive and differentially regulated genes that take part in oxidative stress response. We propose to assay the survival of these mutants exposed to H2O2under microaerobic conditions and compare that to wild type L. reuteri. Determining the role of specific genes in oxidative stress resistance inL. reuteriwill help provide insight into its anti-inflammatory activities.

Keely Pohl
SIBS Mentor: Trent Tipple (ttipple@peds.uab.edu)
Cell Number Decreases with Suppressors of Site IQElectron Leak and Suppressors of Site IIIQo Electron Leak in Hyperoxia Exposure of Murine Epithelial Cells

Background: Hyperoxia is a key contributor to the development of bronchopulmonary dysplasia (BPD) in premature infants. Bronchopulmonary dysplasia is a form of chronic lung disease in which the lung and bronchi are damaged in the neonatal period and causes dysplasia of the alveoli. Compared to the relatively hypoxic in uteroenvironment, postnatal oxygen toxicity is further exacerbated by the administration of therapeutic oxygen. Hyperoxic exposure enhances cellular oxidative stress; however, the intracellular sources of excess reactive oxygen intermediates (ROI) are poorly defined. Mitochondrial superoxide (O2•-) and hydrogen peroxide (H2O2) are ROI’s produced as a consequence of electron leak during the reduction of molecular oxygen by oxidative phosphorylation. The primary sites electron leak include complex I (site IQ) and complex III (site IIIQo). Suppressors of site IQelectron leak (S1QEL) and site IIIQoelectron leak (S3QEL) have been recently developed to decrease O2•-production without affecting oxidative phosphorylation.
Hypothesis: These studies tested the hypothesis that S1QEL and S3QEL will attenuate the effects of hyperoxia exposure on cell proliferation.
Methods: Immortalized lung airway epithelial cells, a cell type known to be oxidatively damaged by hyperoxia, were usedforthese studies. Mouse transformed club cells (mtCC) wereexposed to hyperoxia (85% O2) or normoxia (21% O2), and H2O2and cell production wereassessed at 1, 2, 4, 6, 24, and 48 hours of exposure to determine the time at which cell count is maximally produced. A heme peroxidase-dependent fluorescent assay wasutilized to quantitate H2O2production. In a separate set of studies, mtCCs weretreated with S1QEL or S3QEL prior to hyperoxic exposure and cell numberswerequantitatedat the time point identified in the above studies.
Results: Hyperoxic exposure decreased mtCC cell number. S1QEL and S3QEL failed to prevent hyperoxia-induced decreases in cell number. S1QEL, but not S3QEL, decreased cell number in both hyperoxia and normoxiawhen compared to the control cells.
Conclusions: Neither S1QEL nor S3QEL improved hyperoxia-induced decreases in mtCC cell number. Surprisingly, S1QEL decreased cell number in normoxic cells. Given the lack of a comparable effect of S3QEL, our novel datareveal that complex one derived O2•-is a key driver of mtCC proliferation. To our knowledge, this unique contribution of mitochondrially derived oxidants in lung epithelial proliferation has not been reported.

Allison Raymundo
SIBS Mentor: Jianhu Zhang (jianhuazhang@uabmc.edu)
Autophagy in Aging and Alzheimer’s Disease

Introduction: Autophagy is responsible for maintaininghomeostasis within the cell byrecycling cellular debris, including degraded organelles and superfluous proteins. Efficiency of autophagy is suspected to decreasewith ageallowing excess proteins and damaged organelles to collect within the cells leading to the development of protein aggregates anddecreasedorganelle quality. Additionally, dysfunction or insufficient levelof autophagy hasbeen linked to Alzheimer’s disease (AD), however, there aremany unknown factors regardingthis relationship.

Objectives: The purpose of this study is to examine the difference in the levels of autophagy in young and old mouse models and wild type (WT) and TgF344-AD ratmodels.

Methods: Western blotting was conducted on mouse and rat model tissue samples to measure their quantities ofLC3 and p62, key proteins in the autophagy pathway.

Results: Young and old mouse hippocampi showed significant differences in their levels of autophagy proteins. The difference in the levels ofLC3-I between the WT and AD rats was insignificant. However, there was a significant difference in the levels of LC3-I between the male and female WT rats.

Conclusions: The higher level of p62 in the older mice could indicate a decreased level of autophagy. However, the insignificance of the ratio of LC3-II/LC3-I does not support that assumption. No conclusions can be drawn regarding the levels of autophagy in the WT and AD rats as the difference was insignificant. However, the difference between the male and female WT rats could be tied to the higher prevalence of AD in women.

Christian Sidebottom
SIBS Mentor: Mark Bevensee (bevensee@uab.edu)
Myofiber Type Proportion Disparity Between Races in Patients with Spinal Cord Injury

INTRODUCTION: Compared with Caucasian Americans (CA), African Americans (AA) exhibit much lower levels of insulin sensitivity, resulting in greater risk of metabolic and cardiovascular dysfunction. Relative proportions of myofibers, broadly classified by myosin heavychain phenotype (MHC I, MHCIIa, and MHCIIx), have been associated with insulin sensitivity in several previous studies; however, research investigating the link between race and muscle myosin heavy chain phenotype is limited among individuals with spinal cord injury (SCI).

OBJECTIVE: The goal for this study is to evaluate differences in muscle fiber distribution between African Americans and their Caucasian counterparts.

METHODS: Myofiber distributions were determined via immunohistochemistry staining performed on flash frozen cross sections obtained from the left vastus lateralis via muscle biopsy in 20 adults with SCI without type II diabetes.

RESULTS: There were a total of 20 (13 AA, 7 CA) patient samples used in this study. The highest proportion of fiber types in both groups was MHCIIa (AA: 55.7 %; CA: 53.1%), however, no statistically significant differences were found in any of the three myofibers between AA and CA.

CONCLUSION: Our data suggests racial differences in people suffering from spinal cord injury does not affect the muscle fiber distribution in the paralytic vastus lateralis muscle. In future studies, larger samples sizes should be used to provide more statistical power to the data. Analyses should also include age as a factor for consideration to expand the scope of the study. Future research should also explore the relationship between insulin sensitivity and myosin heavy chain type in patients with SCI.

Ashley Smith
SIBS Mentor: Martin Young (martinyoung@uabmc.edu)
REV-ERBα Influences on Autophagy in WT and CBK Hearts

Introduction: Research evidence suggeststhat circadian clock gene BMAL1is acritical regulator of metabolism,protein production, gene expression, etc. in mammalian cells. When BMAL1 is deleted or dysfunctional, chronic hypertrophyoccurs in the heart.Ineffective autophagy, the primary mechanism of marking and digesting cellular debris, over time decreases the functionality of the heart. mTOR functions tofacilitateprotein synthesis, cell growth, and cell survival.Transcription factor REV-ERBα is also regulated by BMAL1, but its impact withintheheartis not well understood.

Objectives: We hypothesizethat REV-ERBα affects autophagyin the heart.In ourpresentstudy, we investigated the effects ofincreasing REV-ERBαactivityusing of a selective agonist (SR9009)in cardiomyocyte-specific BMAL1 deficientKnockout(CBK) and wild type mice modelson cellular autophagy.

Methods: CBK and wild typemice were injected with the agonistor control solution intraperitoneally(100mg/kg/day,7 days), after which whole hearts werecollect for assessmentof phospho-mTOR(p-mTOR), totalmTOR(t-mTOR), andautophagy markerLC3IIprotein levelsusing western blot.

Results: There were nostatisticallysignificant changes inLC3II, p-mTOR, or t-mTORbetween the 4 experimental groups.However,agonist increased p-mTOR independent of genotype. Levels of t-mTOR and L3CII remained unchangedin WT mice. CBK mice protein levels were affected independent of protein, with a slight decrease in LC3II and slight increase in t-mTOR.

Conclusions: Based onthese results, these results suggestREV-ERBα mayinfluencemTOR activity. Whether REV-ERBαinfluences autophagy or protein synthesis(processes whichmTORcontrols) require further investigation.

Hannah Stumpf
SIBS Mentor: Sooryanarayana Varambally (soorya@uab.edu)

INTRODUCTION: Although well studied, breast invasive carcinoma(BrCa) isone of the top causes for cancer-related deathsin women.Cancer initiation,unregulatedgrowth,invasion, andmetastasiscan be characterizedbycomplexmolecularevents. Genomic dysregulations may play a role in forming these characteristics. Onegene,sperm-associatedantigen5 (SPAG5),was found to be upregulated in aggressive breast cancer.

OBJECTIVES:The main objective of our study wasto corroborateThe Cancer Genome Atlas (TCGA) data showing SPAG5 overexpression in BrCa and to explore the potential of SPAG5 as a targetable oncogene.

METHODS: Using the UALCAN database, The TCGA RNA-sequencing data of patients was analyzed. Immunoblots were used to compare SPAG5 protein expression between multiple BrCacell lines and anormal breast cell line, HMEC. Finally,using a small cohort of patient samples, immunohistochemistry (IHC)was performed for SPAG5.

RESULTS: RNA-sequencing data was analyzed for SPAG5 by comparing transcript abundance between cancer stage and subclassin which SPAG5 expression was statistically significant when compared to normal breast (p < 0.0001). Three BrCacell lines(MCF-7, BT-474, and MDA-MB-231) were compared to normal breast cells and analyzed via western blot, with β-actin as a control.The relative density of the band darkness correlated with the previous RNA-sequencing data. One slide in our cohort of patient tissues strongly supports SPAG5 expression in breast cancer tumorsfor IHCanalysis.

CONCLUSION: Our preliminary results shows overexpression of SPAG5 in breast cancer. In future, we will assess the significance of SPAG5 in breast cancer cell growth, proliferation and tumor progression.

Brielle Tobin
SIBS Mentor: Peter King (phking@uabmc.edu)
FRZB is a novel potential biomarker for Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a terminal neurodegenerative disease of the upper and lower motor neurons whose symptoms manifest as peripheral muscle weakness and muscle atrophy. It progressively spreads centrally, eventually affecting the diaphragm and causing difficulty breathing. Only 5-10% of ALS cases are familial and diagnoses are only made once symptoms arise. Thus, a biomarker is necessary for presymptomatic diagnosis, which may lead to potential early treatment before degeneration occurs. RNA sequencing data identified elevated Frizzled Related Protein (FRZB) expression in the muscle of SOD1G93Amice,a murine model for ALS. FRZB is an intriguing target as a marker for ALS because it is a secreted protein, and might be detectable blood which is preferable to a muscle biopsy.qRT-PCR data has validated elevated FRZB mRNA in ALS patient muscle samples aswell as in the ALS mouse model, and immunohistochemistry has indicated FRZB protein overexpression in ALS patient muscle. These results demonstrate the possibility ofFRZB as an idealbiomarker for ALS.

Kaeli Zoretich
SIBS Mentor: Ravia Bhatia (rbhatia@uabmc.edu)
Preliminary Phenotypic Characterization of Human Bone Marrow Mesenchymal Stromal Cells from Normal and Leukemic Samples

Introduction:Chronic myeloid leukemia (CML) isa type of blood cancer that ischallenging to completely treat. Someleukemic stem cells are able to evade therapy-induced apoptosis,andmesenchymal stem cells (MSCs) areimplicated in this problem. However, it is not understood how MSCsare altered in CML.

Objectives: The goalof this project was tocharacterize the phenotypic differences between normal and CML patient-derived MSC samples, andto assess their differentiation ability.

Methods: Bone marrow samples from normal andCML patients were processed to isolate MSCs in culture. Samples were differentiated into adipose, chondrocytes, and osteocytes and stained for analysis. Immunophenotypingwas used to characterize differences between MSC samples.

Results: MSCswere isolated from 3normal and 5 CML bone marrow samples. CML MSCs took much longer to grow in culture. Two normal MSC samples were differentiated successfully. Osteocyte growth was similar between both normal samples, but adipose growth was varied.Staining to confirm chondrocyte identity was unsuccessful. Flow cytometry data of MSCs indicated that CML samples have significantly less MSCs (indicated by CD271+/CD140a-), and that this population of cells may be in a more primitive state (indicated by TNAP-)and designatedfor osteogenic differentiation (indicated by CD51+).

Conclusion: MSCs can be successfully differentiated into adipose and osteocytes, but more work needs to be done to achieve chondrocyte differentiation. As expected, MSCs isolated from normal and CML samples differ in their proportions of measured cell surface markers. This preliminary characterization needs to be repeated with larger sample sizes and more precisely controlled variables to make more confidant conclusions about CML MSCs.

SIBS Student: Lexis Deshazor-Burnett
SIBS Mentor: Yabing Chen (ybchen@uab.edu)
The Role of ATG7 in Vascular Smooth Muscle Cell Calcification

Vascular Calcification is prevalent in atherosclerosis, renal failure, and diabetes. Vascular smooth muscle cell calcification, a process that mimics the formation of bone, stiffens vascular tissues and inhibits contraction. Autophagy has been shown to have a role in vascular calcification particularly in stressful conditions such as low potassium. Elevation of autophagy has beenlinked to increased calcification. Several genes have been linked to regulation autophagy including ATG7 which has been shown to have an essential role in autophagic flux. We sought to investigate the role of autophagy in calcification of vascular smooth muscle cells in ATG7-knockout mouse models. Knockout of ATG7 globally has been shown to be fatal,and the Cre/lox system has been used to generate tissue-specific knockouts. The presence of calcification following exposure to environments containing sodium phosphates and hydrogen peroxide using ex vivo organ culture of aortic rings and in vitro vascular smooth muscle cells was examined using alizarin red staining and quantification of cell calcium content. Expression of cell markers, including Runx2 and ɑ-SMA, and ATG7 and GAPDH, in calcification of vascular cells and transition to a bone-like structure was also assessed. ATG7-knockout cells were found to exhibit less expression of the Runx2 osteoblast differentiation marker, and less calcium content than the ATG7 f/f cells. The results suggest that cells deficient in ATG7 do not undergo calcification at levels similar to cells that have not lost essential autophagy genes.

SIBS Student: Hannah Douglas
SIBS Mentor: Ganesh Halade (ganeshhalade@uabmc.edu)
The Deficiency of ALOX5 Leads to Amplified Cardiorenal Inflammation In Acute Heart Failure Post-Myocardial Infarction

Introduction: Renal inflammation is obviousprofoundafter myocardial infarction (MI; heart attack) in progressive heart failure (HF). In renal inflammation, the role of ALOX has remained undetermined.
Objectives: To determine the role of ALOX5 after MI-induced HFand renal inflammation and test whether the absence of ALOX5 facilitatesresolving or non-resolving inflammation.
Methods:Ischemic HF and inflammatory responses in Wild Type and ALOX5-/-/-mice were induced via ligation of left anterior descending coronary artery. Acute (days 1-5 post-MI) and chronic (day 56 post-MI)HFtime points were studied and compared to No-MI (day 0) controls. In acute and chronic HF, histology determined cardiac and renal structure and echocardiography measured heart function. Renal biomarkers and molecular gene expression markers were determined.
Results: HF pathology was marked with heart dysfunction and pathological remodelingin comparison to no-MI d0. Post-MI,echocardiography determined that the ejection fraction was decreased in WT and ALOX5-/-compared to no-MI controls. Immune cell density was amplified in acute and chronic HF, particularly in the ALOX5-/-/-group. Lack of ALOX5 magnified inflammation and gene expression markers in acute HF in the ALOX5-/-group. The plasma creatinine levels in ALOX5-/-mice demonstrated a significant declinein renal function measured againsteach respective WT time point. Inthe ALOX5-/-group, ALOX15 and -12, COX1, and nephrin increased on d5 compared to the MI-d5 WT. Measured against MI-d1 WT, the COX2 in ALOX5-/-constantly increased from d1, and Ngal spiked on d1.
Conclusions:The lack of ALOX5 promotes non-resolving cardiorenal inflammation in acute HF.

SIBS Student: Alex Gann
SIBS Mentor: Jennifer Braswell Christy (jbraswell@uab.edu)
Relationship Between Vestibular Dysfunction and Fatigue in Persons with Multiple Sclerosis

Introduction: The vestibular system works in part with the somatosensory system and the ocular system to achieve an optimal state of balance. Multiple Sclerosis (MS) is a neurological disorder that causes damage to the Central Nervous System (CNS) which in turn develops scar tissue around the nerve fibers. Fatigue is one of the most significant defining characteristics of MS.

Objectives: The objective of this study is to test the vestibular function within persons with MS and show the relationship between it and their clinical and subjective level of fatigue.

Methods: This is a case study where each participant completed a subjective Expanded Disability Status Scale (EDSS), Fatigue,and Balance Confidence surveys and underwent clinical testing using the Sensory Organization Test (SOT), Functional Gait Analysis (FGA), and the 6min walk test. The clinical tests were compared both to the normative values and between each participant showing clinical variances caused from different durations of diagnoses.

Results: There were non-significant moderate negative and positive correlations, respectively, between the 6-minute Walk distance and perception of fatigue (R2= -0.40, p=0.50) and SOT performance (R2= 0.80, p=0.20).

Conclusions: Physical fatigue seems to be related to both self-report fatigue and balance. Self-reported fatigue does not seem to have a relationship with measured balance. This discrepancy may be due to the inclusion of mental and social fatigue instead of simply physical fatigue in the surveys.

SIBS Student: Braden Garrison
SIBS Mentor: Namakkal Soorappan Rajasekaran (rajnsr@uab.edu)
Lipid Peroxidation Signaling is Critical for C2C12 Myoblast Differentiation

Oxidative stress can lead to loss of muscle mass and function, which contributes to age-mediated skeletal muscle complications such as sarcopenia, impaired satellite cell regeneration, and reduced energy production. Reactive oxygen species (ROS) play a major role in lipid peroxidation (LPO), and antioxidant consumption in humans is believed to nullify oxidative stress. However, the excess intake of antioxidants may suppressthe basal ROS/lipid peroxidation signaling necessary for basic cellular function, leading to poor regeneration during injury. Here, we hypothesized that a low-dose lipid peroxidation is critical for myoblast differentiation. C2C12 myoblasts were cultured in normal growth medium (proliferation medium –PM), and differentiation was induced by exposing the cells to medium supplemented with 2% horse serum (differentiation medium –DM) and every 48 h (Days 1, 3, and 5), cells were replaced with fresh medium. MTT based assays were performed to analyze the survival rate of cells after treatment with different doses of malondialdehyde (MDA) (0.5 to 100μM) for 24 hours. Dosages of 0.5 and 1μM were selected to test the effect of MDA on C2C12 differentiation. As shownby assessment of morphology and rating of differentiation, 0.5μM of MDA appeared to significantly inhibit myoblast differentiation compared to the control while 1μM of MDA seemed to only slightly affect differentiation. This unexpected outcome suggests there may be an optimum, pro-oxidative redox state that exists that promotes C2C12 myoblast differentiation.

SIBS Student: Grace Goodall
SIBS Mentor: Ceren Yara (cyarar@uab.edu)
Targeting Skeletal Muscle with Neuromuscular Electrical Stimulation (NMES) to Improve Fiber Type Distribution and Size in Patients with Spinal Cord Injury (SCI)

INTRODUCTION: Within 6 months after spinal cord injury (SCI), lower limb muscles atrophy by up to 45% compared to able-bodied (AB) controls. Moreover, within 6 months after SCI, fatigue-resistant and oxidative Type I and Type IIa muscle fibers transform into highly fatigable, glycolytic Type IIa and IIx muscle fibers with impaired oxidative metabolism.

OBJECTIVES: Assess the effects of combined-NMES on muscle fiber type and cross-sectional area (CSA) in individuals with sub-acute SCI.

METHODS: Six participants (28.3±7.3 years old, SCI C6-L1) with sub-acute SCI were recruited from the UAB Spain Rehabilitation Center and randomly assigned into intervention or control groups. This study combines aerobic NMES and resistance (NMES-RE) training in order to preserve both Types I and IIa muscle fibers and improve muscle mass after SCI. Study participants received NMES-RE and NMES aerobic treatment three times a week. Muscle biopsies of the vastus lateralis muscle were performed pre- and post-intervention.

RESULTS: The Comb-NMES group increased Type I and IIa fiber distribution while increasing Type I CSA by 1% compared to a 11% decrease in the control group. Type IIa and IIx CSA decreased from 5-20.5% in the control and Comb-NMES groups.

CONCLUSIONS: This study is ongoing and results may change with a larger sample size. Our preliminary data suggests that Comb-NMES treatment may increase the size and distribution of Type I and distribution of Type IIa fibers after SCI.

SIBS Student: Courtnee Harpine
SIBS Mentor: Sara J. Gould (sgould@uabmc.edu)
A Retrospective Data Collection of the Internal Fracture Fixation of Long Bones with the Stryker Plating System

Stryker plates and screws are commonly used in individuals to provide stability for the healing of long bone fractures. The performance and safetystandard of careforthe Stryker Plating System (SPS) must be assessed to ensure the product works with limited consequences.The primary objective is to demonstrate the performance and safety of SPS via bone consolidation in correct alignment and reporting of anyadverse effects.This was an observational study that included retrospective chart review of pre-operative, operative/discharge and the standard of care follow-up visit data in individuals who received the SPS. The primary endpoint was determining the performance of the SPS by showing if the bone consolidated in the correct alignment or not through radiographic and/or clinical assessments. An additional endpointincluded accessing the safety of the SPS. Safety was analyzedthrough information on device-related adverse effects. Fifty cases were included. Descriptive statistics showed that theeffectiveness ofSPS at consolidating the bone in correct alignment occurred 94percent of time.Of the six percent that did not experience bone consolidation only one case had revision surgery. The average number of days after surgery that bone consolidation occurred was 145 days. An increase in the number of days until consolidation was seen as the Gustilo-Anderson classification increased.No device related adverse effects were seen. The performance and safety of the SPS was determined in this study based on fifty cases.Largercomparative studies need to be performed to determine the performance and safety on a larger scale.

SIBS Student: Minhee Jeon
SIBS Mentor: John J. Shaka (johnshacka@uabmc.edu)
Exploring the AutophagyLysosome Pathway forPotential Biomarkersof Neurocognitive Impairment in HIV

HIV has become a chronic disease due to the advent and success of antiretroviral therapy (ART), resultingin relatively normal lifespans. Consequently,several age-related comorbidities are occurring at higher prevalence in people living with HIV (PLWH), despite ART-mediated control of viral suppression. Our lab is interested in delineating the pathogenesis and treatment of HIV-associated neurocognitive disorders (HAND). Previous studies have indicated alterations in theautophagy-lysosome pathway (ALP), an enzymatic degradation pathway,in peripheral blood mononuclear cells (PBMCs) in HIV infectionsetting. However, whether the function of the ALP is altered in PBMCs with respect to neurocognitive status or ART has not been investigated. Wehypothesizethat ALP function is altered in PBMCs from PLWH, effects that are further exacerbated by cognitive impairment and minimized by ART. Wefocused on monocytes, a cell type with known involvement in CNS HIV infection. We isolated CD14+ monocytes from PBMCs obtained from the Aids Clinical Trials Group (ACTG)repository from cognitively normal PLWH or PLWH with mild cognitive decline, either before or 48 weeks after ARTand from healthy controls (HCs). Monocytes were treated in the presence or absence of chloroquine, a lysosome inhibitor that in turn inhibits the ALP. The status of autophagic flux was assessed through western blot analysis of LC3-II (specific marker of autophagosomes) and P62 (macroautophagy substrate) that accumulate with chloroquine treatment. Ongoing analysis of western blot data from these experiments will indicate whether autophagic flux is altered in monocytes as a function of HIV infection, cognitive decline and ART.

SIBS Student: Carolyn Kittrell
SIBS Mentor: James J Kobie (jjkobie@uabmc.edu)
The Influence of CD4+ T Cell Depletion on the Development of HIV-1 Envelope Specific Antibodies

In order to develop an effective vaccine for HIV, a B cell response must be induced which produces persistent broadly neutralizing antibodies. No vaccine thus far has been able to achieve a sustained and diverse humoral immune response to a variety of HIV isolates. Although the central focus of past investigations has been on the roles of IgG and IgA, we propose IgM may be a key player in prevention of HIV infection and should be researched further. In this study, we utilized high-throughput isolation of immunoglobulin genes from HIV envelope (Env) specific single B cells to generate recombinant monoclonal antibodies (mAbs). These cells were taken from two treatment groups of rhesus macaques, one of these groups was depleted of CD4+ T cells prior to vaccine administration and SHIV challenge to generate a more robust IgM response. The resulting monoclonal antibodies were characterized for reactivity by ELISA to determine their breadth and the specific regions of the envelope protein which the antibodies target. We successfully isolated HIV envelope-specific mAbs. Characterization of these mAbs revealed a possible preference for VH4-2 gene usage, and these mAbs target regions of Env including C1. Further characterization of these and other mAbs is expected to determine if CD4+ depletion impacts the B cell response to the HIV vaccine.

SIBS Student: Scotti Lindon
SIBS Mentor: Jianbo Wang (j18wang@uab.edu)
Mapping of the Spatial Pattern of TBX1 and TBX5 to Determine the Cause of Congenital Heart Defects in Mice

Background: Today, scientists have identified that the planar cell polarity pathway(PCP) isintegral in mice heart formation. This pathway helpsdetermine the shape, orientation and morphology of the cardiac progenitorsin the second heart field (SHF),thereby promoting the elongation of the SHF and the deployment of SHF progenitors to both the arterial and venous pole of the developing heart. SHF progenitors fated for the arterial pole express Tbx1, whereas those fated for the venous pole express Tbx5.The objective of this study is to investigate how theexpression pattern of Tbx1 and Tbx5in the SHF may change upon lossof PCP ligand WNt5a.

Methods: E9.5 wild-type and Wnt5a-/-mutantmouse embryos were frozen and then sectioned, immune-stainedfor TBX1 and TBX5.These images were used for 3D reconstruction to determine the expression domain and boundariesof TBX1 and TBX5.

Results: TBX1 and TBX5 showmutually exclusive expression patternsin second heart field in wild-type embryos, while their expression areas overlap in Wnt5a-/-embryos.We anticipate that as a consequence of disrupted morphogenesis in mouse PCP mutants, the fate specification of SHF cells for arterial vs. venous pole will be disrupted.

Conclusion: The mutual exclusive spatial pattern of TBX1 and TBX5 could be crucial for second heart field morphogenesis and development and Wnt5a might be an important regulator for the establishment of such pattern, which in turn could contribute to heart development.Further investigation is needed to fully understand the role of the planar cell polarity pathway with congenital heart defects.

SIBS Student: Natalie Lipari
SIBS Mentor: David Knight (knightdc@uab.edu)
Relationships between White Matter Microstructure,Neurochemistry, and Conditioned Fear Responses in Posttraumatic Stress

Introduction: Posttraumatic Stress Disorder (PTSD) results from experiencing or observing a psychological trauma and is characterized by cognitive-affective dysfunction. PTSD symptoms appear to be mediated by brain regions that include the prefrontal cortex (PFC), amygdala, hippocampus, and hypothalamus. These brain regions support emotional learning, expression, and regulation processes that appear to be disrupted by PTSD. Further, several white matter pathways connect these brain regions, including the cingulum bundle,uncinate fasciculus, stria terminalis, and fornix. The structural variability of these brain regions and the white matter that connects them may be linked to the cognitive-affective dysfunction associated with PTSD. Using neuroimaging and psychophysiologicalmeasures, we examined the relationship between white matter structureand conditioned fear responses in both trauma-exposed and non-trauma exposed participants.

Objectives: The presentstudy investigatedwhether the white matter microstructureof the brain relates to the emotional response produced during Pavlovian fear conditioning. Abetter understanding of the relationship between white matter and fear responses may provide novel insight into thecognitive-affective dysfunction linked toPTSD. In addition, magnetic resonance spectroscopy (MRS)wasused to study whether neurochemicals within the dorsal anterior cingulate cortex (dACC)vary with the conditioned fear response.PTSD patients show hyperactivity within the dACC, however theneurochemicalprocesses that underlie dACC hyperactivity have yet to be determined.

Methods: Twenty-one trauma exposed (TE) and nineteen non-trauma exposed(NTE)individuals participated in this study. Participantscompleted a magnetic resonance imagingsession during whichdiffusion weightedand MRSimages were collected. Neuroimaging was completed approximatelyone month post-traumafor TE participants. Seventy-two conditioning trials were presented during the study. Three types of conditioning trialswere presented (CS+, CS-, and UCS alone)in a pseudorandom order, ensuring that no two trial types werepresented consecutively. Using amagnetic resonance imaging (MRI)compatible joystick, participants continuously rated their expectancy of UCS presentation on a scale of 0 (certain the UCS would not be presented) to 100 (certain the UCS would be presented). A physiological monitoring system was also used during the MRI scanto measure skin conductance responses(SCR).

Results: The white mater microstructure of the left cingulum bundleand left stria terminalis/fornixwas positively correlated with expectancy of the UCS alone in the NTE group. The left stria terminalis/fornix showed a positive relationship with SCRto theUCS alonein the TE group. The TE group showed negativecorrelations between white matter microstructure and neurochemicals. Specifically, the white matter of the right uncinate fasciculusvaried with Glx(glutamate and glutamine)concentrations. Moreover, thewhite matter of theright stria terminalis/fornixshowed a negative relationship with N-acetyl-aspartate levels. Anegative relationship was observed between the white matter microstructure of the left uncinate fasciculus and creatine, while a positive relationship was observed between the white matter ofthe stria terminalis/fornixand Glx (glutamate and glutamine) in the NTE group.Finally, the NTE group showed several positive relationships between neurochemicals and SCR. SCRto the CS+UCS (i.e. the UCSthat followed the CS+)increased as Glx and choline concentrations increased.

Conclusion: The present findings suggest that the neurochemistry and white matter microstructure of the human brain influencethe emotional response produced during Pavlovian fear conditioning. Findings from this project provide new insight into the relationship between white matter, neurochemicals, posttraumatic stress, and Pavlovian fear conditioning processes.Future studies should look further into the role theseneurochemicals and white matter pathways play in both healthyand dysfunctional emotional processes.

SIBS Student: Ronisha McCardell
SIBS Mentor: Serra A. Rosa (rserra@uab.edu)

Chronic back pain due to intervertebral disc (IVD) degeneration is an aging disease that will affect 80 percent of adults. The degeneration of the IVD, more specifically the fibro-cartilage of the annulus fibrosus, is the cause of the pain. During spinal development, TGFß(transforming growth factor beta) signals in the sclerotome, which is a tissue composed of mesenchymal stem cells, to regulate the formation of the annulus fibrosus. It has been established in mice that TGFßis an important signal needed to form the annulus fibrosus, but the mechanism is unknown. Scleraxis isa transcription factor that is regulated by TGFßsignaling during spinal development. We hypothesize that Scleraxis is an important signal regulated by TGFßsignaling tomake the annulus fibrosus. To test this hypothesis, we inhibited Scleraxis mRNA using siRNA in C3H10T1/2cells and inhibited TGFßsignaling using a synthetic inhibitor in chick embryos. By inhibiting TGFβ signaling and most importantly Scleraxis, we expect to see a down regulation in annulus fibrosus tissue markers, ADAMTSL2 and Fibromodulin, if Scleraxis is an important signal required for annulus fibrosus formation. If our hypothesis iscorrect, then TGFßand Scleraxis could potentially be used in stem cell therapies to treat chronic back pain and other spinal disorders.

SIBS Student: Meredith McCloy
SIBS Mentor: Yacoubian, Talene (tyacoubian@uabmc.edu)
Modulated Levels of 14-3-3θ Protein Activity and Regulation of Pathogenic α-Synuclein in Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder hallmarked by neuronal cell loss and the formation of proteinaceous aggregates in the brain. Alpha-synuclein (α-syn) is the key protein composing these inclusions. Misfolded α-syn is thought to propagate in a prion-like fashion by templating the aggregation of endogenous native α-syn. There are currently no disease modifying therapies for PD. Therapeutic targeting of the modifiers promoting α-syn aggregation could lead to disease modifying therapies. 14-3-3 proteins are chaperone proteins with high neuronal expression which have cellular functions affecting protein trafficking, neurite growth, and cell death. Dysfunction of 14-3-3 has been implicated in α-syn pathology. Using the in vitro pre-formed fibril (PFF) model, we have previously published that 14-3-3θ reduces α-syn spread and toxicity, while inhibition of 14-3-3s accelerates the spread and toxicity of α-syn. This suggests that 14-3-3θ may also be protective in vivo. Here we used the α-syn PFF in vivo model to test the impact of 14-3-3s in a more biologically relevant system. Wefound that 14-3-3θ overexpression decreased α-syn aggregation at 3mpi but increased aggregation at 6mpi. This may indicate that disease progression was delayed and peak aggregation was shifted to a later time point with 14-3-3θ overexpression. Conversely,14-3-3 inhibition increased α-syn at 3mpi but decreased aggregation at 6mpi. It has previously been published that cell loss follows α-syn aggregation in the substantia-nigra. We are currently investigating the impacts of 14-3-3θoverexpression or inhibition on synaptic loss and cell death in the cortex by NeuN cell counts and synapsin staining.

SIBS Student: Chelsey Norwood
SIBS Mentor: Karen Gamble (klgamble@uab.edu)
Investigation of amyloid beta plaque accumulation in the hippocampiof transgenic mice

INTRODUCTION: Alzheimer’s disease(AD)is the most prevalent cause of dementia worldwide and is characterized by pathological amyloid beta (Aβ) plaques and tau tangles.Subclinical epileptiform activity or seizures, which are indicative of network hyperexcitability, are also present in early Alzheimer’s disease.Interestingly, incidence of epileptiform activity and seizures is greater during the night and early morning hours in otherwise healthy people andis increasedin ADpatients. This could be exacerbated due to disruptions in their sleep-wake cycle, manifested as fragmented sleep-wake activity patterns, suggesting a disrupted circadian clock. One cause of this hyperexcitability could be a decrease in inhibitory activity. Preliminary data from our lab show increased day-time inhibition compared to night-time onto CA1 pyramidal cells (PCs) of thehippocampus of wild-type mice. This increased day-time inhibition is lost in our AD mouse model, the Tg-SwDI. Loss of inhibition could lead to increased excitation of PCs, and hyperexcitability, as seen in other AD mouse models, including the J20,which overexpresses human amyloid precursor protein (hAPP) with the Swedish and Indiana mutations. In addition, increased neuronal activity leads to increased Aβplaque accumulation. The J20 mouse model has impaired cognitive function by 4 months andexhibits plaque pathology later. Going forward, we will use this AD model,as the delay in cognitive decline and plaque accumulation are important for future experiments. First, it is important to examine when pathology first appearsin J20 mice. We hypothesized that 4-month-old hAPP (+) J20 mice will have Aβ plaque accumulation in contrast tothose that are hAPP (-). Our results showed no Aβplaque accumulation in 4-month J20 mice or their littermate controls. We will also examine plaque accumulation at 6 and 8 months. Wealso hypothesize that increasing inhibition will delay the onset of plaque pathology. Therefore, future experiments will use chemogenetics targeting interneurons to increase inhibition todelay the onset of plaque pathology and cognitive impairment in the J20 mouse model.

OBJECTIVE: To determine if loss of day-time inhibition contributesto cognitive impairment and Aβplaque accumulation in Alzheimer’s disease patients.METHODS:To investigatethe presence of Aβpathology in hAPP J20 mice,we used intracardial perfusions with phosphate buffered saline and paraformaldehydeto preserve mouse brains, sectioned brains using a microtome, stained for beta pleated sheets as a proxy for mature amyloid beta plaques using ThioflavinS, and imaged tissue with a fluorescence microscope.We specifically imaged tissue containing the hippocampus, our brain region of interest. Future experiments will involve stereotactic surgery to inject AAV-DREADDs and behavioral assays and ELISAs and immunohistochemistry to examine cognitive function and amyloid pathology, respectively.

RESULTS: Mature Aβplaques were not present in the hippocampus of 4-month J20 mice or their littermate controls via ThioflavinS staining.

CONCLUSIONS: ThioflavinS staining was successful as seen in control, 12-month hAPP(+) J20 mice. At age 4-months, J20 mice do not exhibit mature Aβplaques. Future experiments will be performed to examine plaque accumulation of 6-and 8-month mice.

Keywords: Alzheimer's disease, amyloid beta, circadian, hippocampus, J20

SIBS Student: Austin Primeaux
SIBS Mentor: Bin Ren (bren@uabmc.edu)
FoxO1-Activated CD36 Transcription Switches Arteriolar Differentiation of Endothelial Cells

Introduction:Transcription factor FoxO1 is the most physiologically critical regulator of endothelial stability, essential for vascular homeostasis via its modulation of downstream genes important for angiogenesis and vascular maturation. FoxO1 has been shown to promote expression of vascular endothelial growth factor. Unfortunately, the mechanisms by which FoxO1 specify the different cellular responses and gene targets in endothelial cells (ECs) have not been fully elucidated. The CD36 gene contains FoxO1 binding sitesthat control its transcription. CD36 is an angiogenic regulator and fatty acid receptor. The LPA/PKD-1 signaling inhibits CD36 transcriptional expression by regulating FoxO1 activities and chromatin remodeling, and mediates transcriptional reprogramming of microvascular ECs (MVECs) for proarteriogenic responses. Objectives: We aimed to test thehypothesisthat FoxO1 initiates an arteriolar switch that regulates the development of the microvasculature via regulation of CD36 transcription. Methods: To test this hypothesis, we transduced FoxO1 into late passages of primary human MVECs by lentiviral infection and observed arteriogenic gene expression. Results: We showed that forced expression of FoxO1 significantly increased CD36 mRNA levels by qPCR. FoxO1-induced CD36 expression was attenuated by co-transduction of FCoR, a specific FoxO1 co-repressor. LPA exposure also inhibited FoxO1-induced mRNA expression of CD36. Intriguingly, FoxO1 transduction stimulated the expression of ephrin B2 and Hey2 in primary HUVECs. Conclusion: Our studies suggest that FoxO1 doesinitiate an MVEC arteriolar switch likely via promoting increases in CD36-mediated fatty acid metabolism and VEGF signaling, thus enhancing functional angiogenesis.

SIBS Student: Alleyah Ransom
SIBS Mentor: Kristen Allen-Watts (krallen@uab.edu)
Examining the Use of Complementary and Integrative Health Approaches among African American and White Adults with Heart Failure in the Deep South

Due to the increasing popularity of complementary and integrative health (CIH) in Western nations, researchin this field has followed suit. Overall, most studies done on the subject report most CIH users to be Caucasian, upper class, female, and having higher educational attainment. However, because these studies tend to lack culturally sensitive CIH approaches, not much is known about CIH use among various racial and ethnic groups, especially those residing in the deep South. The purpose of this study was to examine the use of CIH approaches in the past 12 months among African American (AA) and White (W) patients with heart failure in the deep South; identify the reported reasons for using CIH approaches among AA and W patients with HF in the deep South; and to examine patient disclosure to physicians about the use of CIH approaches among AA and W patients with HF in the deep South. This investigation was done through the use of secondary data analysis from a study including responses from 250 HF patients from various ethnic groups. The results found that more Ws used CIH in the past 12 months than AAs. Additionally, more W’s reported using CIH for pain relief while more AAs reported using CIH for overall wellness. Finally, more AA patients disclosed their use of CIH to clinicians than W patients.

SIBS Student: Ashlyn Scheinost
SIBS Mentor: Li Y Li (liyli@uabmc.edu)
The Relationship Between Adolescent Obesity and Depression

Background:Childhood obesity and depression are both universal health problems that affect millions of Americans today. The prevalence of both diseases would insinuate that they co-occur, but limited research has been conducted to determine if they are functionally related. This study is designed to establish the correlation between adolescents with major depressive disorder and obesity.

Methods: Seventy-five subjects participated and completed this study for data analysis. TheMini International Neuropsychiatric Interview (MINI) was conducted to assess the mental health in the participants,and the participants were divided into two groups: major depressive group (MDD) and control group. The Quick Inventory of Depressive Symptomatology (QIDS) was used to measure the severity of depression. The Adverse Childhood Experience (ACE) questionnaire was given to assess for early life stress. Fat composition was measured by dual-energy X-ray absorptiometry. Physical activity was determined by the Physical Activity Questionnaire. Diet was determined by the Brief Eating Assessment for Pediatric Patients (BEAPP).

Results: There were no significant differences in the physical activity and diet score between participants in the MDD group and non-MDD (control) group. However, there are significant differences in the fat total, trunk fat, fat percentage, and the composition of fat between the two groups.

Conclusions: Our data indicated that there is a relationship between adolescent obesity and MDD independent of physical activity and diet. Further research regarding the psychological correlation to obesity could aid in developing tailored treatments to help decrease the rates of both obesity and MDD in adolescents simultaneously.Keywords: adolescent, obesity, depression

SIBS Student: Evan Suppa
SIBS Mentor: Kirk Habegger (kirkhabegger@uabmc.edu)
The Role of beta-Hydroxybutyrate in Glucagon Receptor Stimulated Food Intake Suppression in DIO mice

INTRODUCTION: Glucagon’s counterregulatory role to insulin action is well known, but its broader therapeutic potential is an evolving research interest. We have reported that chronic glucagon receptor (GCGR) activation decreases body weight (BW) and food intake (FI) while increasingbeta-Hydroxybutyrate(HB). Recentstudies suggest thatketone esters similarly reduce food intake in mice.

OBJECTIVES: The primary objective of this study was to determine ifdecreases in FIseen in diet induced obesemice treated with GCGR agonist (IUB288) are due to production of HB.

METHODS: C57Bl6/J micewerefed High Fat Diet (HFD, 58% fat + sucrose) for 12 weeks,to stimulate DIO.Micewere assigned into groupsmatched for food intake;Vehicle-Vehicle, Vehicle-IUB288, Trimetazidine-Vehicle, Trimetazidine-IUB288,(n=3). Micewere treated with or without Trimetazidine, 15 mg/kg (TMZ), an inhibitor of ketogenesis, for two days prior to four daysofIUB288 or vehicle injections. Following aten-daywash out period, the study was repeatedusing30 mg/kg. FIand BW weremeasured daily,andplasmaHBwere measured at baseline and study conclusion.

RESULTS: IUB288 reduced FI and BW in both TMZ and vehicle treated mice.Surprisingly HBconcentration was elevated post-treatment in both 15 mg/kg and 30 mg/kg TMZ treated mice.

CONCLUSION: In both experiments, the administered dosage of TMZwas insufficientto blockIUB288-stimulated HB.The results provide neitherevidence for, oragainst, HBFI suppression in DIO mice.Future studies will utilizea larger dosage of TMZ, or administration of a different inhibitor of ketogenesis.

SIBS Student: Dana (Mackensie) Terry
SIBS Mentor: Jane Allendorfer (allendjb@uab.edu)
Preliminary study of physical activity levels, gray matter volumeand verbal memoryin people with idiopathic generalized epilepsy

People with epilepsy are less likely to engage in physical activity than healthy controls and half report cognitive deficits. Physical activitymay benefitcognitive function. In a preliminary cross-sectional study, we investigate the relationship between hippocampal grey matter volume (GMV), memory function, and physical exercise in leisure (PEL) in persons with idiopathic generalized epilepsy (IGE) and healthy controls (HC). We hypothesize positive relationships betweenPEL,memory function,and hippocampal GMV.

Adults with an EEG-confirmed IGE diagnosis (N=19) and age-matched HC(N=12) completed the Baecke Questionnaire to assess PEL.Participants performed the Hopkins Verbal Learning Test (HVLT) (n=24) or the California Verbal Learning Test (CVLT-II)(n=8) function; z-scores for Total Recall, Delayed Recall, and Recognitionassessed verbal memory function. Participants underwent a 3T MRI; voxel-based morphometry (VBM) assessed GMV.

Two-sampled t-testsshowed both groups had similar verbal memory and GMV(p>0.05), though PEL was greater for HC than IGE (p=0.005). Multiple regression analyses showed PELdid not predict memory scores for either group, but PEL did predict GMV in left and right hippocampi for IGE (p=0.036 and p=0.033, respectively) but not for HC (p>0.05). Left hippocampal GMV predicted Total Recall (p=0.033) and Recognition (p=0.033) in IGE, while no relationship was found in HC (p=0.592 and p=0.929, respectively).

PEL predicted hippocampal GMV but not memory function in IGE, although hippocampal GMV predictedmemory function. Studies need to include larger numbers of IGE and HC participants to fully explorethese relationships.