Changes in transforming growth factor- b signaling and activation with stretching of mouse lung epithelial cells

SIBS student - Emily Courtman

SIBS mentor - Namasivayam Ambalavanan M.D.

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).

A yeast model for development of CF therapeutics

SIBS student - BreeOna Ebrecht

SIBS mentor - John Hartman

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.

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

SIBS student - Joshua M. Freda

SIBS mentor - Robert A. Kesterson

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.

Novel Mechanisms for the Treatment of Breast Cancer: In Vitro and in Vivo Models

SIBS student - Rebecca Garrett

SIBS mentor - Boris Pasche

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.

Determining the Presence and Localization of Septin2 and Tulp1 in Rod Cells

SIBS student - Meredith Hubbard

SIBS mentor - Alecia Gross

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.

Characterization of the periplasmic ferric binding protein of Mycobacterium smegmatis

SIBS student – Andrea Loes

SIBS mentor – Michael Niederweis

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.

The Effects of Green Tea Component, EGCG, on Triple Negative Breast Cancer Cells

SIBS student - QueenDenise Okeke

SIBS mentor - Trygve O. Tollefsbol

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.

Engineered polycaprolactone and cellulose substrates for MCF-7 F-108 co-cultures

SIBS student – Jeremy Shepard

SIBS mentor – Joel Berry

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.

5-Hydroxytryptamine 1B Receptor (5-HT1B) expression in the human caudal brainstem in major depression

SIBS student - Aneesh Tyle

SIBS mentor - Ilan Kerman

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.