How Ironic: A Novel Approach to Stabilizing Influenza-Induced Pulmonary Inflammation
The influenza virus increases inflammation to the lungs, which can lead to secondary complications such as pneumonia. Iron oxide nanoparticles may prove to be a more effective treatment than existing therapies. Use of such nanoparticles has been increasing in the medical field due to their potential for targeted delivery of drugs. Nanoparticles have been demonstrated in recent studies to stabilize induced inflammation in other cell lines. However, other studies have reported that iron oxide nanoparticles are toxic to certain cells lines. This study measured the inflammation of lung cells through the application of tumor necrosis factor alpha (TNF-α), an inflammation inducing protein that is secreted after the influenza virus enters the epithelial cell. Levels of nitric oxide were used as the index of inflammation. It was hypothesized that cells stimulated with TNF-α would produce higher levels of nitric oxide than stimulated cells with the application of iron oxide nanoparticles. Results showed that cells stimulated with TNF-α in the presence of low concentrations of iron oxide nanoparticles produced nitric oxide levels that indicated decreased cellular inflammation (p<0.05). Cell viability was used to measure toxicity. Increased levels of iron oxide nanoparticles resulted in a higher percent of cell viability and upregulated cell growth (p<0.05). Future studies will need to determine the optimal concentration of iron oxide nanoparticles that will stabilize cell inflammation while simultaneously promoting cell growth, leading to more effective strategies for targeted influenza therapies in lung cells.
Linking Diabetes to Alzheimer's Disease in Neuroblastoma Cells via TGFbeta1
Alzheimer's Disease (AD), the leading cause of dementia in elderly people, is frequently associated with amyloid beta (Abeta) plaques in the brain, and an increased ratio of Abeta42/Abeta40 isomers has been connected with an increased prevalence of these plaques. Type 2 diabetes mellitus (T2D), a failure to properly respond to insulin, has been noted to increase the risk of AD; although this connection is not yet fully understood, TGFbeta1, a cytokine that regulates a variety of cellular functions, is suspected to be heavily involved. The purpose of this experiment is to identify the role of TGFbeta1 in increasing the Abeta42/Abeta40 ratio in BE2-M17 neuroblastoma cells under T2D conditions. It was hypothesized that if under T2D conditions, the BE2-M17 cells would yield increased Abeta42/Abeta40 ratio and TGFbeta1 levels. Additionally, it was hypothesized that they would yield a normal Abeta42/Abeta40 ratio even under T2D conditions if the TGFbeta1 signaling pathway is inhibited. The ELISA results for Abeta40 and 42 levels report that variations in glucose and insulin exposure have no significant effect on Abeta40 levels, but decreases glucose, insulin, and insulin-like growth factor 1 levels significantly increases Abeta42 levels. This suggests that a combination of hypoglycemia and hypoinsulinemia increases the risk of AD.
Jenna DiRito & Serena Tharakan
Nanoparticle-Mediated Gene Delivery via Balloon Angioplasty to Suppress Intimal Hyperplasia
In the U.S. alone, over 200,000 cardiovascular surgical procedures requiring grafts fail annually, primarily due to restenosis caused by the physiological healing response known as intimal hyperplasia (IH). IH can be attributed to endothelial cell proliferation, narrowing blood vessel walls through key gene VEGF-A, a gene allowing for the growth of the endothelial cell layer called the intima. Pdx1 is a homeobox whose promoter can possibly induce overexpression of VEGF-A. It was hypothesized that genes that interfere with IH encapsulated within nanoparticles and coated onto a surgical balloon could specifically and efficiently target the region of interest. Such a novel form of genetic delivery may overcome the obstacles of current gene delivery such as non-specificity and inefficiency. shRNA-regulated knockdown of Pdx1 delivered to a rat carotid artery via PLGA nanoparticles, created via the double emulsion method, could lower VEGF-A expression, thereby preventing endothelial growth, the development of a neo-intima and ultimately occlusion. Glycerol, a highly viscous substance, was used to suspend the nanoparticles, allowing adherence onto the surgical balloon. The coated surgical construct was employed in a left carotid artery intimal hyperplasia injury model, performed by the students, to test its efficacy and compared against a standard balloon catheter in the control condition. Fluorescence microscopy was used to detect which cells in the arteries had taken up the anti-Pdx1 plasmid that was also tagged with GFP, confirming the successful delivery of the contents of the nanoparticles. rt-PCR was used in sample analysis to assess the extent of IH and levels of VEGF-A. TEM analysis was also performed to analyze quality of the nanoparticles. Significant results (p < 0.05) support the proposed hypothesis. While intimal hyperplasia was the initial model chosen for study, further research will apply the delivery method to various venous and arterial conditions.
Novel Targets in Atherosclerosis and Lupus Nephritis: Curcumin and Hydroxychloroquine Treatment on Cholesterol-Coding Genes
Chronic inflammatory conditions, such as lupus, have become increasingly prevalent, and frequently affect the kidney. Termed lupus nephritis, this condition features inflammation of the kidney s blood vessels and glomeruli containing cholesterol-laden macrophages. Atherosclerosis also features large accumulations of cholesterol plaque, largely derived from cholesterol-laden macrophages. Research has shown that pro-inflammatory cytokines cause an upregulation of genes that stimulate cholesterol biosynthesis, specifically MVD and DHCR7. The goal of this experiment was to investigate the gene regulating effect of certain substances on MVD and DHCR7, providing a potential salutary effect in the treatment of lupus nephritis and atherosclerosis. Specially, this experiment studied the effects of the pro-inflammatory cytokine IL-6, curcumin and hydroxychloroquine. Curcumin is a popular and natural substance used to treat inflammation, and hydroxychloroquine is a common prescription drug used in the treatment of lupus. Using native cells in media as the control group, the effects of curcumin (up to 60 micromolar), hydroxychloroquine (up to 60 micromolar) and IL-6 (up to 40 nanograms) were studied individually and in combination, examining the effect on gene regulation and, by inference, the formation of cholesterol-laden macrophages. Effects on gene expression were examined through RT-PCR. Cells treated with IL-6 alone demonstrated an upregulation of expression for both MVD and DHCR7 in comparison to the baseline. IL-6 expression increased with increasing concentration, until 40 nanograms of IL-6 were introduced. Gene expression then decreased, but still remained higher than the baseline. My results were as predicted in the hypothesis, except for the change in gene expression with 40 nanograms of IL-6. Furthermore, cells treated with curcumin demonstrated a drastic down-regulation of expression for MVD, also as predicted in the hypothesis. The curcumin levels almost entirely suppressed the expression of MVD in the cells. The extent to which curcumin suppressed the gene expression in MVD was not predicted in my hypothesis. Further results are pending.
Effects of Valeryl Salicylate on Interleukin-4-Mediated Pathways in K-562
Interleukin-4 (IL-4), a cytokine found in high levels in tumor microenvironments, induces cancer cell proliferation through a mechanism yet to be fully elucidated. In vitro research has suggested the IL-4-induced activation of the JAK/STAT6 pathway may contribute to cell proliferation. Interestingly, previous findings show that salicylates inhibit STAT6 activation in murine lymphoma. It was hypothesized that treatments with valeryl salicylate would inhibit IL-4-induced proliferation in chronic myelogenous leukemia cells. K-562 cells were treated with IL-4 (100 ng/mL) for 96 hours. At 72 hours, valeryl salicylate (1-4 mM) was added. At 96 hours, an MTS assay showed that valeryl salicylate (1-4 mM) significantly reduced cell viability (p<.05). STAT6, BCL-2, SHIP-1 and ERK1/2, downstream targets of IL-4 binding, were measured as each are reported to influence cellular proliferation through independent pathways. Results from ELISA demonstrated that valeryl salicylate (4 mM) significantly decreased levels of phosphorylated STAT6 (p<.05). Valeryl salicylate did not significantly decrease levels of BCL-2, SHIP-1 and ERK1/2, suggesting that valeryl salicylate inhibits IL-4-induced proliferation and survival through a pathway independent of these proteins. Therefore, valeryl salicylate may act as a potential therapeutic option in preventing chronic myelogenous leukemia cell proliferation through a STAT6-mediated pathway.
Effect of cTnT on Cardiomyocyte Reprogramming
In 2011, a study from the California Institute of Regenerative Medicine showed how the addition of three different transcription factors (TBX5, GATA4, and Mef-2C) caused fibroblasts to behave like cardiomyocytes, or cardiac muscle cells. This process, in which a type of cell becomes another without a pluripotent step between the two, is known as transdifferentiation. It was observed that cells treated with the three transcription factors showed an increased expression of the cTnT gene, which is known to assist in cardiac muscle contraction. However, a later study at the Boston Cardiovascular Research Center repeated this experiment and found that the method of creating cardiomyocytes was inefficient and that the induced cardiomyocytes (iCMs) the method produced had a decreased rate of cell survival and low expression of cardiomyocyte-specific genes. The objective of this experiment was to investigate the role that cTnT plays efficient reprogramming to iCMs. This was assessed by transforming cells that already exhibit upregulated cTnT and comparing these cells to iCMs without artificially upregulated cTnT. Comparisons suggest that an upregulation of cTnT in iCMs causes decreased expression of other cardiomyocyte specific genes without significantly affecting cell viability. Later experimentation assessed the effects of cTnT upregulation outside of its role in transdifferentiation, in order to determine the mechanisms with which cTnT upregulation interferes. Data suggests that cTnT upregulation interferes with calcium signaling, a vital process in both induced and natural muscle cells.
Naturally Reducing Pancreatic Beta-Cell Deterioration By Controlling Oxidative Stress
This research investigates how cinnamaldehyde (CND), comprising 90% of the Cinnamonum zylancium (cinnamon) essential oil, can reduce intracellular reactive oxygen species (ROS) in pancreatic islet beta-cells and therefore increase insulin secretion of these cells due to minimized inhibition of transcription factors PDX1 and MafA. RIN-5F insulin-secreting pancreatic beta-cells were tested in vitro with CND concentrations 0, 100, 500, 1000, and 1500 micromolar in RPMI 1640 media with 2760 micromolar H2O2 for 24 hours (n=2, n=6). Results showed increased ROS with higher concentrations, statistically significantly different from control at p<0.05 for 500 and 1500 micromolar. Insulin secretion decreased with higher concentrations, statistically significant from control at p<0.05 for all concentrations, and at p<0.01 for 100 micromolar. RIN-5F beta-cells were incubated with 3520 micromolar H2O2 and CND concentrations of 0, 0.01, 1, 10, 100, and 1000 micromolar in RPMI media for 24 and 72 hours (n=3, n=4). For 24 hours, cell viability tests showed increased viability with increasing CND concentrations, statistically significantly from control at p<0.05 for 0.1, 1, 10, and 1000 micromolar, demonstrating a reduction of the effect of H2O2 on the cells. For 72 hours no trend was found. RIN-5F beta-cells were incubated with the same CND concentrations for 48 hours (n=5). Cell viability was most at 0.1 micromolar, statistically significantly different from control at p<0.05. Incubation for experiments was at 37 degrees-Celsius/5%CO2/100% humidity. A beneficial low-dose CND therapeutic effect is strongly indicated. Future research includes CND beta-cell trials with PDX1/MafA silenced, and CND effect on glucagon and somatostatin levels.
Changing Cellular Identity: Direct Cell Reprogramming Using Chromatin Remodeling Complexes
The reprogramming of fibroblasts to create induced pluripotent stem cells (iPSCs), which can then be differentiated into many cell lineages, has been explored. A major obstacle in the use of iPSCs is tumor formation in vivo, thereby dashing any clinical value. The reprogramming of fibroblasts to iPSCs raises the possibility that a somatic cell could be reprogrammed to an alternative differentiated fate without first becoming a stem/progenitor cell. It has been found that fibroblasts can be reprogrammed into cardiomyocytes using a combination of three developmental transcription factors, Gata4, Mef2c, and Tbx5 (iCM factors). In this experiment, it is shown that the combination of the three iCM factors along with two chromatin remodeling complexes (BAF60c and INO80) demonstrates more efficient reprogramming of dermal fibroblasts directly into differentiated cardiomyocyte-like cells. In order to evaluate cardiomyocytic behavior, levels of expression of cardiomyocyte-specific genes ACTC1 and RYR2 and secretion of cytokine IL-6 in cell media, were analyzed by qPCR and ELISA, respectively. Fibroblasts to which the chromatin remodelers were introduced showed a significant increase in ACTC1 and RYR2 expression, as well an increase in IL-6 secretion. These findings demonstrate that chromatin remodeling complexes enhance direct reprogramming of differentiated fibroblasts to cardiomyocyte-like cells. Through using chromatin remodelers, reprogramming of endogenous or explanted fibroblasts might provide a source of cardiomyocytes for regenerative approaches more readily, due to the higher reprogramming efficiency. Having a means of obtaining virtually any cell type from fibroblasts (an abundant cell type), without the risk of tumorigenicity, could lead to the use of cellular reprogramming as regenerative therapies for currently untreatable diseases.
Photosynthetic Algae/Insulinoma Cell Fusion Creating Self-Sustaining Insulin Producer
Insulin is a hormone that regulates blood sugar concentration. In the approximately 300 million diabetic patients worldwide, utilization and production of insulin is substantially and in the case of Diabetes mellitus type I patients, completely impaired. These patients require the direct pumping of exogenous insulin into the blood. Current methods of insulin production are expensive and require complex protocols. This project explores a means of creating an inexpensive, low maintenance cell hybrid that will produce insulin while creating useable chemical energy via photosynthesis. Cells of the photosynthetic green-algae species, Chlorella kessleri, and cells from the rat insulinoma cell line, RIN-5F, are fused to create cell hybrids known in this experiment as Modified Insulin Production (MIP) cells. It is hypothesized that successful fusion of algae and insulinoma cells will lead to the implementation of an efficient, inexpensive approach to in vitro insulin production via a multi-kingdom cell hybrid that contains the biochemical properties of each of its cellular components. Before fusion, the algae cells are relieved of their rigid cell walls in solution containing cell wall-degrading enzymes cellulase and pectinase. Fusion is then stimulated with polyethylene glycol (MW 4000). Ultra-Sensitive Rat Insulin ELISA is performed on the pre-fused insulinoma cells and on the MIP cell hybrids to test for insulin concentration in media. Successful fusion is observed via light microscopy (400x) and scanning electron microscopy (up to 10,000x). Transmission electron microscopy (up to 9,000x) is used to visualize algal cell wall degradation. The algae/insulinoma fusion process represents the potential for the inexpensive production of lifesaving bioproducts by means of multi-faceted cell hybrids. Statistically significant (P<0.05) results of the ELISAs show notable insulin production by the MIP cell fusions (approximately 40 ng/mL). Most recent experimentation includes implementation of fusion between C. keslerri and beta cells cultured directly from rat pancreas. Scanning electron microscopy proves effectiveness of this more intensive fusion procedure. Further assays regarding insulin production by the algae/rat pancreatic cell hybrids are pending.
An Experimental Investigation of neurological functions of MECP2 Protein
MeCP2, a relatively unexplored gene, is known to regulate methyl CpG protein 2 (pMeCP2) that is important in the proper function of nerve cells. The mutation of MeCP2 has led to cases of Rett syndrome, a little known genetic disorder. Parkinson s disease, a more widely known disorder is also caused by genetic mutation, which results in cognitive decline and seizures. Both disorders share comparable symptoms, as well as similarities on a genetic level. MeCP2 is already known to affect the methylation of cytosines and in turn, possibly regulate cell growth and development. With MeCP2 mutated, cell death or stunted cell growth is prevalent. In Rett syndrome patients, this is evident specifically in the substantial nigra area of the brain where dopaminergic cells are concentrated. After transfecting neuroblastoma cells (of the cells line Be(2)-M17) and silencing the MeCp2 gene, it became clear that cell growth was impaired. Cells began to grow sparse fragments and die quickly. Recent research has suggested that dopamine, as well as the neurotransmitters, serotonin and norepinephrine, have the ability to induce pMeCp2 (protein regulated by the MeCP2 gene). Rett syndrome patients lack sufficient pMeCP2 production could therefore be linked to lack of proper dopamine production by MeCP2 deficient neurons. Parkinson s disease which is caused by PARK1 mutations, also results in significant decrease in dopamine secretion and significant cell death. Information about Rett Syndrome could be relevant to Parkinson s disease and vice versa, because both diseases are linked to significant decrease in dopamine secretion and cell death. Because these two aspects of the disease are the same, scientists can now begin to think about other links between the two disease and ways of linking treatments. Furthermore, future plans are to treat both groups of cells with trans-retinoic acid that could not only help suppress the cell death rate, but help increase dopamine production and regulate GABA production. (Data Pending)
Control of Induced Pluripotent Stem Cell Aging by Modulation of Mitochondrial DNA Deletions
Induced pluripotent stem cells (iPSCs) could revolutionize patient-specific regenerative medicine, but their premature aging symptoms currently limit their clinical applicability. In this study, it was hypothesized that this phenomenon is triggered by the age-related mitochondrial DNA (mtDNA) common deletion, and that by modulating mitochondrial genomic damage, rapid aging in iPSCs and other disorders could be controlled. Healthy cells were reprogrammed into iPSCs by three different methods. The common deletion was induced in all cells, suggesting that the mtDNA defect is technique independent. Further disparities were observed in mitochondrial size and morphology between iPSCs and their source cells, changes paralleling those seen in cancer cells. Although derived from older patients, the cancer cells tested did not exhibit the age-related deletion, suggesting a unique pathway of mtDNA conservation, thought to be mediated by Sirtuin 1 (SIRT1), a gene regulating nuclear / mitochondrial cross-talk. Both knockdown and inhibition of SIRT1 induced the common deletion in cancer cells, selectively inducing senescence. Conversely, SIRT1 activation in iPSCs ameliorated aging features, suggesting methods to maintain clinically useful iPSCs without rapid aging. Importantly, these effects were absent in patient cells containing maternally-inherited mtDNA deletions, suggesting that while genetically-inherited mitochondrial defects may be irreversible, age-related mitochondrial features can indeed be ameliorated. Bioinformatic analysis identified a number of genes that could be developed into mitochondrial genome controllers for other age-related diseases such as Parkinson s disease, Alzheimer s disease, amyotrophic lateral sclerosis, fibromyalgia, and diabetes.
Novel Methods to Eliminate Senescent Cells and Cancer
In humans, the aging process has been implicated in a wide variety of diseases including cancer, cardiovascular disease, alzheimer s disease and diabetes mellitus type II. However, it has been found that the elimination of senescent cells in mice with the premature-aging disease progeria reduces the extent of aging symptoms present. Furthermore, senescent cells have been found in aging human tissue where they display a phenotype linked to cancer and increased inflammation. The chemical Torin 2 acts mainly by inhibiting the mTOR (mechanistic target of rapamycin) pathway in human cells, and Resveratrol acts primarily via the activation of SIRT1. The purpose of this research was to test the ability of these chemicals to inhibit or reverse the senescent phenotypes of Human Adult Dermal Fibroblasts (HADF). The experiment involved adding varying doses of Torin 2 to fibroblasts in vitro. Then, after 4 days, a $beta$ galactosidase (SA-$beta$ gal) senescence assay was performed and an analysis via microscopy revealed a slight decrease in senescence with added Torin 2, as compared to a control group with no Torin 2 added. Experiments with Resveratrol were inconclusive. However, Torin 2 microscopy results provide further evidence of the potential clinical benefits of Torin 2 and mTOR inhibition for combating senescence in humans.
Vitamin K and Dopachrome Tautomerase: Aids to Chemotherapy in Melanoma
Melanoma is an aggressive form of skin cancer known for its resistance to chemotherapy. Even though the detrimental effects of cancer are clearly visible on the patients, the molecular changes behind those effects are not. Research has shown that melanoma cells have elevated levels of DOPAchrome Tautomerase (DCT). Melanoma cells also produce large quantities of Reactive Oxygen Species (ROS). Vitamin K3, a lipophilic vitamin, has been shown to decrease the creation of ROS, leading to the hypothesis that K3 might work in conjunction with DCT reduction to enhance the effect of drug-induced killing. Cisplatin (CDDP), one of the severest chemotherapeutic drugs currently being used in patients, works by directly damaging DNA molecules. While all three methods of treatment are individually effective, it was hypothesized that their combination would be more deadly to the cancerous cells. In order to test this, DCT was down-regulated with an shRNA plasmid and its nonsense plasmid. Concentrations of Vitamin K3 and CDDP we added over 24 hours, during which the most significant decrease in cell viability was expected. The percent of successfully transfected cells, and the percent viable cells were counted using flow cytometry post transfection. The data showed that the combination of CDDP and Vitamin K3 application on un-transfected cells is the most effective in initiating cell death. A killing curve conducted with non-cancerous cells also indicates that aforementioned treatment would be less-likely to harm non-cancerous cells.
K-Ras as a Novel Target in Glioblastoma Multiforme Treatment
Glioblastoma Multiforme (GBM) is the most common brain tumor in adults characterized by aggressiveness, invasiveness and rapid proliferation. Most patients diagnosed survive no longer than a year. GBM s rapidity of proliferation makes it very difficult to eradicate since it continues growing before, during and after treatment. In GBM, aberrant signaling in the Ras/Raf/MAPK pathway leads to the proliferation and maintenance of the cancer. KRAS is an important gene in this pathway that activates further downstream factors including phosphorylated ERK, and finally leads to cell proliferation. This research investigated the effects of K-Ras inhibition on the proliferation and on the activity of phospho-ERK in the GBM cell line Ln229. Small hairpin RNA (shRNA) plasmids with green fluorescent protein (GFP) were used to inhibit KRAS expression in vitro. Proliferation was analyzed by comparing percentages of transfected versus non-transfected cells overtime. Phospho-ERK levels were analyzed using an enzyme linked immunosorbent assay (ELISA). Data obtained showed no significant decrease in levels of phospho-ERK after KRAS inhibition. This indicates that simply inhibiting KRAS is not sufficient to halt activation of this pathway. Data on proliferation is pending.
Cell Suicide: MDM2 Knockdown Promotes Apoptosis Via Reactive Oxygen Species
Reactive oxygen species (ROS) are very reactive molecules. These molecules can cause significant damage to cellular structures. Both ROS-elevation and ROS-elimination strategies have been generated to induce cell death in cancerous cells. This project focuses on ROS-elevation due to the fact that noncancerous cells are known to have a higher survival rate with higher concentrations of ROS than cancerous cells. The modulation of ROS concentrations does not seem to be an ideal therapeutic strategy to induce apoptosis in cancer cells due to the adaptations to ROS stress by the cancer cells. However, this project couples the regulation of ROS concentrations with the knockdown of MDM2 to selectively induce apoptosis in cancer cells. MDM2 is a negative regulator of the p53 tumor suppressor. Overexpression or amplification of this locus has been detected in a variety of different cancers. In normal cells, the activation of p53 induces cell cycle arrest, while in some cancerous cells, the activation of p53 induces cell cycle arrest and apoptosis. Decreasing the expression of MDM2 by gene knockdown decreases the chance of survival for the cancer cells. The A549 non-small lung cancer cell line was transfected with MDM2 shRNA plasmid, which generates a small hairpin RNA, which then silences target gene expression via RNAi (RNA interference). Apoptosis was then induced by a range of different concentrations of hydrogen peroxide (H2O2). The data collected thus far shows that inducing apoptosis with hydrogen peroxide as well as knocking down MDM2 increases the percent of early apoptotic cells and decreases the percent viability. This approach could be extended to clinical use, using small molecule inhibitors of MDM2 in place of shRNA. MDM2 inhibitors will activate p53 in both cancerous and noncancerous cells. More data pending.
Nicholas Haas & Matthew Korst
Measuring Blood Flow in Arteries
Artery bypass surgery is a procedure used to help maintain blood flow through an artery when blockage is present. In the procedure, an incision is made in the artery and a graft is attached to circumvent the blockage. We experimented to determine whether a circular or slit incision is the more efficient method. A tubing circuit was created to mimic the artery and the joint where the experimental artery connected with the experimental graft was placed under a light microscope. A 40% glycerol solution was made to imitate the viscosity of blood. When experimenting with various amounts of blockage, the flow through the circular incision went straight through. In the slit, a small bubble formed and the flow rotated upon entering the graft. It can therefore be concluded that the circular incision is more efficient than the slit incision.