Health Sciences 2017

Health Sciences Index by Poster #:

1. Development of a high output experimental model to study drug resistance in cancer |  Steven Wu, Chayanin Kiratipaiboon and Yon Rojanasakul 

   Department of Pharmaceutical Sciences, West Virginia University, Morgantown, WV 26506 

   Chemoresistance is one of the major obstacles to successful cancer therapy. ABCG2, a transport protein, has been reported to play a critical role in chemoresistance. Emerging evidence indicates an overexpression of ABCG2 in cisplatin treated non-small cell lung cancer (NSCLC) leading to cisplatin resistance. However, there is a lack of information on the causal relationship between ABCG2 expression and cisplatin resistance as well as the underlying mechanisms in NSCLC. This study aims to develop a rapid and quantitative experimental model to investigate the casual relationship between cisplatin-induced ABCG2 expression and chemoresistance in NSCLC. Using ABCG2-luciferase (LUC) reporter H460 cells as a model system, cisplatin resistance was induced and IC50 values of cisplatin and luciferase activity of cisplatin-resistant cells were determined by cell viability using Graphpad Prism and by luciferase assay, respectively. The results demonstrated a linear correlation between luciferase activity and IC50 in cisplatin-resistant lung cancer H460 cells (r2 = 0.83). This finding may facilitate the development of a high throughput screen for anticancer therapeutics and for new drug discovery and mechanistic investigations. 

2. A role for tumor macrophage-derived microvesicles in regulation of epithelial-to-mesenchymal transition |  Kaltume Usman Ndayako, Amy Gross, Joyce Obeng, Clay Marsh, Timothy D. Eubank and Duaa Dakhlallah 

   Microbiology, Immunology & Cell Biology, School of Medicine, WVU Cancer Institute, West Virginia University, Morgantown, WV 26506; Davis Heart and Lung Research Institute, School of Medicine, The Ohio State University, Columbus, OH and Science and Medical Biotechnology, Department of Health Science, Universita Del Piemonte Orientale, Novara, Italy. 

   Macrophages are classified into two populations: M1, pro-inflammatory and anti-tumor to support cytotoxic T cell function, and M2 which suppress immune cell killing and support angiogenesis and progression. Genes involved in Epithelial-to-Mesenchymal transition (EMT) regulate tumor cell plasticity and dictate metastasis while GRHL transcription factors suppress EMT. We investigated a role for macrophage-secreted microvesicles (MVs) to modulate EMT. Our data suggests that MVs derived from M1 and M2 macrophages are taken-up and disparately induce GRHL1/2 mRNAs expression in mammary epithelial and breast cancer cells. Surprisingly, we found that M2 MVs are protective against EMT in mammary epithelial cells by augmenting GRHL1/2 expression and suppressing cell migration, but not protective in breast tumor cells where it was M1 MVs which played the suppressive role. These data suggest that M2 macrophages may play a protective role against initiation of EMT while M1 macrophages may induce reversal of EMT in established breast tumors. 

3. Refining a model for a novel drug treatment of glioblastoma multiforme |  Osasenaga Benjy-osarenkhoe, Neal Shah, Paul R Lockman, Bjorn CG Soderberg, John B Barnett 

   Department of Microbiology, Immunology, and Cell Biology, Department of Pharmaceutical Sciences, and C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506 

   Glioblastoma multiforme (GBM) are stage IV brain tumors with a five-year survival rate of less than 5%. Currently treatments for GBM are not curative and all have potentially devastating side effects. Our lab has been exploring the anti-tumor characteristics of N-methyl-3-(3,4-dichloroanilino)-3-oxopropanoic acid (N-MeDCOPA). N-MeDCOPA inhibits Orai calcium channels, which GBM depend on to increase gene expression. Due to promising inhibition in vitro, an in vivo model must now be developed. U251MG glioblastoma cells were transfected to induce luminescence prior to intracranial implantation into a nude mouse model. Imaging indicated that the tumors were growing, although all mice survived 30 days with no treatment. This demonstrated a need for fine-tuning of the methods before moving forward. Because this compound had not been tested in an animal model of GBM, we explored the pharmacokinetic profile and have developed the administration protocol best suited for this project. It was determined that polyethyleneglycol 400 or beta-Cyclodextrin would be used as a vehicle in future experiments. This refined protocol will be implemented in future experiments to determine the treatment potential. 

4. Assessment of inflammation induced by multi-walled carbon nanotubes and carbon nanofibers from U.S. facilities |  Sarah Foster, Kelly E. Smith-Fraser, Vamsi K. Kodali, Tracy Eye and Aaron Erdely 

   West Virginia University, Morgantown, WV 26506 
   Health Effects Laboratory Division, NIOSH, Morgantown, WV 26505 

   Carbon nanotubes and nanofibers CNT/F are engineered nanoparticles used in electronic, medical, and composite industries. The primary risk of exposure to these nanomaterials occurs during dry powder handling after production. Preliminary research reported CNT/F induce pulmonary inflammation. However, CNT/F represent a broad class of materials and minimal research has been conducted on the majority of these materials. Our goal was to test the pulmonary inflammatory response induced by 9 different CNT/F in mice at 1, 7, 28, and 84 days following exposure by oropharyngeal aspiration to 4 or 40g of CNT/F. Materials with larger diameters induced a greater inflammatory cell infiltration, primarily polymorphonuclear cells by 37%, and gene and protein expression (e.g., IL-1β, CCL22/MDC, IL-6) compared to materials with smaller diameters. Also, inflammation was sustained longer in larger diameter materials with resolution by 84d. Our findings support that CNT/F may be stratified by physicochemical characteristics. 

   Disclaimer: The findings and conclusions in this report are those of the authors and do not represent the views of the National Institute for Occupational Safety and Health. 

5. Community assessment and education to promote behavioral health planning and evaluation (CAPE) |  Rachel A. Wattick and Melissa D. Olfert 

   Division of Animal and Nutritional Sciences, Davis College of Agriculture, Natural Resources & Design, West Virginia University, Morgantown, WV 26506 

   Mental health and substance use disorders affect approximately 20% of the U.S. population and are often comorbid. Clinicians and behavioral healthcare workers report lack of resources available to guide their programs and too great of a gap between research findings and program implementation. CAPE was created to address this discrepancy between evidence obtained from research and program dissemination and implementation. Phase I compiled behavioral health data from ten communities into easy to read snapshots and reports. Phase II created affordable, replicable, and adaptable community-level behavioral health interventions. Index and Innovation interventions included Mental Health First Aid Training, Mindfulness Training, and creation of an addiction awareness website, AddictionHappens.org. The results of this initiative were implementation of evidence-based practices and compilation of results for other communities to easily replicate. Next steps focus on addressing mental health and substance use disorder stigma and implementing a holistic, psychosocial approach to treatment, as patients have reported a three-fold preference for this over medication-based treatments. Overall implication is improvement in treatment outcomes by empowering individuals to drive and sustain their recovery. 

6. Use of metal organic frameworks for drug delivery systems |  Aishwarya S. Vijay, Alixandra L. Wagner and Cerasela Zoica Dinu 

   Department of Chemical & Biomedical Engineering, West Virginia University, Morgantown, WV 26506 

   Metal Organic Frameworks (MOFs) are hybrid materials consisting of metal ions and organic bridging molecules that form networks of empty pores. Studies showed that their internal surface area is large enough to be used for the slow-release of encapsulated medications delivered to the human body. This research focuses on evaluating the potential of user-synthesized, aluminum-edged MOFs to deliver the cardiac glycoside drug digitoxin to human epithelial cancer cells. Digitoxin was used as a drug model since growing evidence indicates that it is a promising anticancer agent at therapeutic concentrations. Experiments performed using cellular and microscopy assays evaluated cell ability to uptake MOFs and determined the dose that leads to maximum cell viability. Furthermore, spectroscopical assays evaluated the ability of MOFs to encapsulate digitoxin, while cellular assays characterized the cell fate upon delivery of MOF-digitoxin complexes. Preliminary results showed that cell ability to uptake MOFs or complexes is drug-dose dependent and hint at the potential of MOFs to serve as the next generation of potent drug delivery vehicles that ensure high drug loadings under versatile functionality. 

7. Patient and nurse perspectives on safety screening in emergency department and urgent care settings |  Lauren Dirkman and Danielle Davidov 

   Department of Emergency Medicine, West Virginia University, Morgantown, WV 26506 

   The inclusion of questions assessing patient safety has become common practice during healthcare visits. However, current research has not shown that safety screening (e.g., about intimate partner violence) is effective in obtaining positive disclosures. There is a need to determine patient and provider perspectives on asking and being asked safety-screening questions. The purpose of this study was to examine the practices and opinions of both nurses and patients in an emergency department and urgent care/student health settings using an anonymous, self-administered questionnaire. Data will be analyzed to determine the types of questions most commonly asked and opinions on comfortability and appropriateness of asking these questions in clinical settings. The results will yield meaningful information about what, if any, changes are necessary to better suit the needs of patients and providers in these settings. This study is significant because to our knowledge, a similar study does not exist and it could be used to improve safety-screening practices. 

8. Quantifying percent breakdown from JWH-018 and heroin to thermal degradation products using peak intensities |  Victoria R. Zeger, Stephen Raso and Suzanne Bell 

   C. Eugene Bennett Department of Chemistry and Department of Forensics and Investigative Science, West Virginia University, Morgantown, WV 26506-6045 

   Novel psychoactive substances (NPSs), such as synthetic cannabinoids, and opiates are frequently ingested by smoking. In literature reports from the 1970s, smoking heroin correlated to increased instances of brain encephalopathies and current abuse of NPSs represents a similar threat to public health. The goal of this project was to exhaustively characterize thermal degradation products of synthetic cannabinoids on plant matter and to revisit by-products of heroin to update the literature. A key objective was to establish percent breakdown of parent compounds to by-products through typical preparation and smoking scenarios and to determine percent recovery of the method used. Samples were collected using a previously designed method and data was analyzed via GC/MS. Mass recovery and percent breakdown of an NPS were observed at approximately 63% and 42% respectively, corresponding to the amount of drug and by-products ingested. Noteworthy results are expected with the analysis of heroin as well. To our knowledge, this is the first study to estimate percent breakdown associated with smoked drugs of abuse, which has numerous clinical and public health implications. 

9. Inhibition of mitochondrial respiration in Acute Lymphoblastic Leukemia within the bone marrow microenvironment |  Patrick M. Thomas, Rajesh R. Nair, Debra Piktel, Werner Geldenhuys, John Hollander and Laura F. Gibson 

   West Virginia University Cancer Institute, Morgantown, WV 26506 

   The current anti-leukemic drugs are unable to induce cell death in the leukemic cells residing with the bone marrow microenvironment, resulting in disease relapse in patients. To counter this hurdle, we have developed a co-culture model that mimics the bone marrow microenvironment and characterized the leukemic cell population that are resistant to drug therapy. Our experimental observations show that these resistant cells have deregulated mitochondrial respiration, which is accompanied by increased activation of hexokinase II and protein kinase B (Akt) and a reciprocal inhibition of AMPK. Interestingly, exposing the co-culture to an anthelmintic drug, Pyrivinium Pamoate, a known inhibitor of mitochondrial respiration leads to cell death in the resistant leukemic population. This Pyrivimium Pamoate-mediated cell death was accompanied by a significant decrease in the ATP production in these cells. Taken together, this study highlights the value of targeting the mitochondria to eradicate drug resistant leukemic population from the bone marrow and thus preventing disease relapse. 

10. Metabolite analyses of rat plasma samples upon exposure to TiO 2 nanoparticles |  Elinore Loch, Sandra Majuta, Timothy R. Nurkiewicz and Stephen J. Valentine 

    C. Eugene Bennett Department of Chemistry and Department of Physiology, Pharmacology, and Neuroscience, School of Medicine, West Virginia University, Morgantown, WV 26506 

    Titanium dioxide (TiO2) nanoparticles are commonly used in a wide range of products, including cosmetics, foods, dyes, and paints. However, research has found that pulmonary exposure to TiO2 nanoparticles may have adverse effects on microvasculature function. Using ion mobility, mass spectrometry and gas-phase hydrogen-deuterium exchange, we are examining whether pulmonary exposure to TiO2 nanoparticles affects circulating metabolites found in plasma. Methanol and chloroform were used to extract polar and nonpolar metabolites from rats exposed to TiO2 nanoparticles by inhalation as well as control organisms. Metabolite extracts were then analyzed on a home-built ion mobility instrument coupled to a mass spectrometer. Metabolite ions were exposed to deuterium oxide in the gas phase and allowed to exchange hydrogen atoms for deuterium atoms. The overall goal was to increase the number of analytical measurements that could be used to distinguish isobaric and isomeric metabolites. A combination of in-house and commercial software used to extract the raw data, align the dataset peaks, normalize the data, and perform principal component analysis (PCA). Metabolites exhibiting the greatest difference between sample cohorts are being submitted to biopathway analysis. 

11. The influence of sex chromosome-specific gut microbiomes on a sexually dimorphic immune response |  Andrea Pettit, Rosana Schafer, Evelyn Onga, Kathryn Blethen and Jennifer Franko 

    Department of Microbiology, Immunology & Cell Biology, West Virginia School of Medicine, Morgantown, WV 26506 and Universidade Federal de Viçosa, Viçosa - MG, 36570-900, Brazil 

    While immune-related sexual dimorphisms have previously been attributed to differences in sex hormones, XX vs. XY sex chromosome complements and distinct gut microbiomes may play a role. In an effort to identify underlying mechanisms contributing to distinct male vs. female immune responses, the gut microbiome composition of four-core genotype (4CG) mice and its influence on a sexually dimorphic immune response was evaluated. 16s rDNA sequencing and metagenomics analysis was performed on DNA isolated from the fecal pellets of 6-8 week old 4CG mice and again 4 weeks post-gonadectomy. Distinct populations of bacteria were identified between XX females and XY males, and also between XX and XY males, suggesting a role for sex chromosome complements in the regulation of gut microbiome composition. Elimination of gut bacteria by antibiotic administration demonstrated a potential role for sex chromosome-specific gut microbiomes on the XX-dependent propanil-mediated enhancement of heat-killed Streptococcus pneunomiae immune responses. By determining mechanisms underlying sexually dimorphic immune responses, gender tailored treatments can be developed to counteract both female-biased autoimmune diseases and decreased immunization responses in males. 

12. Screening method for dietary supplements using direct analysis in real time mass spectrometry |  Emily Gleco, Maíra Kerpel dos Santos, Luis Arroyo and Glen Jackson 

    Department of Forensic and Investigative Science, West Virginia University, Morgantown, WV 26506 

    Some dietary supplements have been found to contain stimulants that are prohibited by the U.S. Food and Drug Administration, World Anti-Doping Agency, and Brazil’s National Health Surveillance Agency for causing adverse side effects, such as cardiac arrest. For crime labs to prove that seized supplements contain these compounds, simple, sensible, and reliable methods are necessary to perform their analysis. Direct analysis in real time mass spectrometry (DART-MS/MS) is being used to develop a faster alternative to current screening methods since it takes only a few seconds to analyze each sample. 109 supplements provided by the Federal Police in Brazil will be analyzed to detect whether 1,3-dimethylamylamine, caffeine, synephrine, ephedrine, sibutramine, and methylphenidate are present. The optimization of DART-MS/MS parameters along with preliminary results obtained by testing standard solutions of these compounds indicate that they can be detected using the developed method. The results of this study will be compared with those obtained by a confirmatory technique using liquid chromatography mass spectrometry (LC-MS/MS). 

13. Toxicity studies of metallic organic frameworks (MOFs) in human lung epithelial cells |  Anna C Eden, Alixandra Wagner, Yon Rojanasakul, Cerasela Zoica Dinu 

    Department of Chemical and Biomedical Engineering and Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, WV 26506 

    Metallic organic frameworks (MOFs) are crystalline-hybrid porous materials with ultrahigh surface areas used in a variety of manufacturing processes, from separation membranes to catalysis and drug delivery. However, the safety of these nanomaterials for humans and the environment is a matter of serious concern. In this study, novel, rectangular box-shaped MOFs were investigated for cytotoxicity in model human lung epithelial cells. For this, cells were exposed to a range of doses of MOFs to determine the IC50, which is the concentration of MOFs where the number of viable cells is reduced by half. Cellular viability was also qualitatively measured after 24, 48, and 72 hours, as well as stress exhibited via reactive oxygen species released by cells following MOF treatment. Morphological evaluation was performed using optical microscopy and real-time via electric cell impedance sensing (ECIS) following exposure. Our analyses have shown that cellular exposure causes cellular fate changes in a dose-dependent manner, supporting the hypothesis that more comprehensive studies on possible health risks of these emerging materials must be performed before use in practical applications. 

14. Development of a cell-based screening platform for drug discovery of neuroinflammation treatment |  Courtney D. Amend, Kelly L. Monaghan and Edwin C.K. Wan 

    Department of Microbiology, Immunology, and Cell Biology, Center for Basic and Translational Stroke Research, Center for Neurodegenerative Diseases, Blanchette Rockefeller Neurosciences Institute,  West Virginia University School of Medicine, Morgantown, WV 26506 

    Transcription factor STAT5 is a potential therapeutic target as its persistent activation has been linked to inflammatory diseases. However, complete blockage of STAT5 activity may result in serious side effects due to its broad physiological roles. Therefore, novel strategies that selectively reduce STAT5 activity are in need. Activated STAT5 forms dimers and tetramers, and we discovered that STAT5 tetramers are critical for the progression of neuroinflammation. Thus, small molecules that reduce tetramer activity may become novel therapeutics for treating neuroinflammation with high selectivity. The overall goal of this project is to develop a cell-based, high throughput platform for screening small molecules that target STAT5 tetramers but not dimers. This goal can be achieved by establishing a cell-based, luciferase reporter assay. To date, we have identified cell lines that can be used for establishing the reporter assay. We are in the process of cloning the luciferase reporter constructs, and herein present the experimental strategies and current progress. 

15. TGF-ß influences proliferation of MDA-MB-231-BR3 triple negative breast cancer cells |  Janelle Chuah, Samuel A. Sprowls and Paul R. Lockman
    
    Department of Pharmaceutical Sciences, West Virginia University, Morgantown, WV 26506- 6045 

    A common phenotype of cancer cells is their unregulated cell growth. Transforming growth factor-β (TGF-β) is a protein that triggers signal pathways crucial for multiple processes such as proliferation, apoptosis, and migration. TGF-ß is implicated in multiple pathways within metastasis of cancer. To demonstrate the effects of TGF-ß on our brain seeding variant of breast cancer cells, a scratch assay was performed using our MDA-MB-231-BR3 (TNBC) cell line. We observed cell proliferation over a period of 24 hours in six different conditions. Conditions included control (media alone), 5ng/mL TGF-ß, 10ng/mL TGF-ß, 1μM SB431542 (TGF-ß Receptor inhibitor), 5ng/mL TGF-ß + 1μM SB431542, and 10ng/mL TGF-ß + 1μM SB431542. Results indicated an increase in percent wound closure when compared to control for both concentrations of TGF-ß. However, the combination approach yielded an increase in percent wound closure as well. A higher concentration of inhibitor may need to be used in future attempts. In conclusion TGF-ß drives proliferation of a brain seeking breast cancer cell line. 

16. Revisiting cortactin as a driver of collective invasion in 11q13 amplified HNSCC | River A. Hames, Brenen W. Papenberg, Jessica L. Allen, Steven M. Markwell, Elyse L. Walk, Amanda G. Ammer and Scott A. Weed 
    
    Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, West Virginia University Cancer Institute, West Virginia University, Morgantown, WV, 26506 
    
    Head and neck squamous cell carcinoma (HNSCC) is an invasive cancer with a five-year survival of 50%. Incidence is increasing Appalachia due to tobacco and HPV exposure. Genomic instability due to carcinogens results in amplification of chromosome 11q13 in 25% of cases. This results in poorer prognosis. The CTTN locus resides in 11q13 and encodes the protein cortactin. Increased cortactin drives invasiveness through cell migration and invadopodia. Invadopodia are membrane protrusions responsible for degrading stromal proteins. Prior studies demonstrated that reduced cortactin expression impairs invadopodia. However, 3D in vitro and knockout mouse models indicates that cortactin removal fails to block invasion. The purpose of this study is to determine the role of cortactin in invadopodia function and invasion. The hypothesis is that HNSCC has compensatory cortactin and/or invadopodia-independent mechanisms capable of driving invasion. We produced cortactin knock-out cell lines with CRISPR-Cas9. These lines will be evaluated for invadopodia function and invasion. Defining the role of cortactin is critical to understanding the consequences of amplification, allowing for intervention and target design in this patient subset.