Skip Navigation

Delta Omega

2014 Poster Competition Winners

Applied Science:

1st Place - Alyssa Frazee

Title: Ballgown: A general statistical framework for RNA transcript assemblies

RNA sequencing (RNAseq) is now the most popular technology for quantifying gene expression. Computational biologists have developed fast, deterministic algorithms that use RNAseq data to reconstruct whole transcriptomes and measure each of the transcripts' abundances in a cell population. However, rigorously testing whether transcripts are differentially expressed between populations remains challenging. Here we propose a general statistical framework, Ballgown, for analyzing the variation in a transcriptome assembly and for detecting differential expression at the transcript, exon, or gene levels. This framework can be used in conjunction with any assembly tool that constructs transcripts and estimates their associated abundances. As compared to the widelyused differential expression package Cuffdiff 2, Ballgown is more accurate for twoclass differential expression testing, flexible enough to handle a wider variety of experimental designs, and computationally more efficient. The Ballgown R package, which currently has builtin support for the popular Cufflinks assembly software, is freely available for download from GitHub.

2nd Place - Katherine Moon

Title: Secondhand Smoke Exposure in Water Pipe Venues in Turkey, Russia, and Egypt: Study Design and Preliminary Results

Despite the successes of smoke-free legislation, tobacco control efforts largely exempt water pipe venues. These exemptions are related to the pervasive belief that water pipe smoking is less harmful than cigarettes. On the contrary, laboratory studies suggest that water pipe secondhand smoke contains increased levels of nicotine, carbon monoxide (CO), and polycyclic aromatic hydrocarbons (PAHs). Our objective was to characterize airborne concentrations of water pipe secondhand smoke contaminants and biomarkers of exposure in venue employees. In 2012-2013, we conducted cross-sectional surveys of water pipe venues and employees in Istanbul, Turkey and Moscow, Russia. Data collection in Cairo, Egypt is planned for December 2013. We enrolled nine water pipe venues (72 employees) in Turkey and 17 venues (104 employees) in Russia. We collected air samples of nicotine, particulate matter (PM2.5), nicotine-derived nitrosamine ketone (NNK), CO, and particle-bound PAHs for at least 24-hours at each venue. Employee exposure was assessed via measurements of hair nicotine, salivary cotinine, and exhaled CO. Urinary NNAL (a NNK metabolite), 1-hydroxy pyrene glucuronide (1-OHPG; a PAH metabolite), and cotinine were also collected. Fieldworkers observed venue characteristics and water pipe smoking behaviors and recorded participant's sociodemographic characteristics and smoking status.

In the nine Istanbul venues, the median (IQR) number of customers served was 120 (100, 200) on weekdays and 350 (200, 400) per day on weekends. 56% of venues had air conditioning and 44% had air conditioning. Although all venues had some anti-smoking policy, a complete indoor ban on cigarette smoking was only in effect in 44%. We detected very high concentrations of CO, PM2.5 and PAHs in water pipe venues. Overall, the median (IQR) levels during of PM2.5, CO, and PAHs during peak hours (between 10 pm and midnight) were 183 (64, 382) µg/m3, 4 (1.5, 23) ppm, and 8 (4, 23) ng/m3, respectively. The overall average concentration of air nicotine was 13.8 (3.3, 14.1) µg/m3. Among venues reporting that 24-49% of their customers smoke water pipe, compared to venues with reporting that 1-24% customers smoke water pipe, the levels of secondhand smoke contaminants were markedly increased, supporting the conclusion that water pipe secondhand smoke is a major source of indoor air pollution in these venues.

We collected biomarkers and questionnaire data from 72 employees. Employees were predominantly male (65%) with a median age of 30 years and low educational attainment (30% less than high school and 58% primary school or less). The employees sampled had a wide range of primary positions (e.g., owner, water/server, bartender), 96% typically worked 6 or 7 days per week, and reported a median (IQR) shift of 10 (9,12) hours per day. 66% of employees reported smoking cigarettes daily and 22% reported smoking water pipe daily. 54% of employees reported that the air-quality at work was either "fair" or "poor". Overall, we found elevated tobacco-specific biomarkers as well as urine 1-OHPG. The median (IQR) urine cotinine, saliva cotinine, and hair nicotine were 465 (378) µg/g creatinine, 235 (16, 670) µg/L, and 11.4 (4.3, 20.6) ng/mg, respectively. The median (IQR) urine 1-OHPG concentration was 0.66 (0.62) µg/g creatinine and the median (IQR) of exhaled CO was 10 (2, 26) ppm. Among non-smokers, 1-OHPG was moderately correlated to urine and saliva cotinine. Among smokers, 1-OHPG was only weakly correlated to urine and saliva cotinine.

3rd Place - Hoda Magid

Title: Compliance with Smoke-Free Tobacco Legislation in Indoor Public Places in 12 Cities in Turkey

The prevalence of cigarette smoking and water pipe smoking was very high among water pipe employees. Their active smoking status combined with their occupational exposure result in extremely high levels of exposure to tobacco smoke, as reflected by very high urine and saliva cotinine concentrations and hair nicotine concentrations. Characterization of water pipe secondhand smoke exposure provides evidence for the inclusion of water pipe venues in smoke-free legislation.

Context: Seconhand smoke exposure poses significant health risks for non-smokers. In 2008, Turkey passed its smoke-free legislation prohibiting smoking in indoor public spaces; but smoke-free legislation is only as effective as its level of compliance.

Objective: To assess the level of compliance with the smoke-free legislation in 12 cities across Turkey (Istanbul, Ankara, Izmir, Adana, Balikesir, Bursa, Erzurum, Gaziantep, Kayseri, Samsun, Trabzon, and Van).

Methods: In each city we selected 10 sampling points using a random sampling strategy. Using a standardized protocol, we visited universities, schools, hospitals, government buildings, shopping malls, and hospitality venues (restaurants, traditional coffee houses, cafes, and bars/nightclubs) between December 2012 and July 2013. During each visit, fieldworkers used checklists to collect information on the number of smokers, the presence of cigarette butts, ashtrays, no smoking signs, fines or penalty signs, signage visibility and cigarette sales. Fieldworkers also observed smoking in taxicabs during rides taken to and from study venues. In each venue, we defined compliance as no smoking observed in any indoor location. In taxis, we defined compliance as no smoking by the taxi driver. We observed a total of 898 venues, 4,395 indoor locations, 39,936 people, and 356 taxi rides. In Istanbul, Ankara, and Izmir, we observed 404 venues, 1,988 indoor locations and 20,120 people. In the 9 smaller cities, we observed 494 venues, 2,407 indoor locations and 19,816 people.

Results: Compliance with the smoke-free legislation was above 95% in schools, 94% in government buildings, 92% shopping malls and 97% in universities. In hospitals, compliance was 79%. In hospitals, schools, government buildings and universities, the areas with poor compliance were the cafeterias and dining areas. In hospitality venues, compliance was 20% in bars and nightclubs, 78% in traditional coffee houses, 93% in restaurants and 94% in cafes. Compliance was 95% in taxis.

Conclusions: Among the 12 cities studied, compliance was high across all cities in Turkey, although it remains below 90% in hospitals and hospitality venues. Moreover, cigarette butts were observed in most locations. These findings support the urgent need to improve the enforcement of the law in hospitality venues, especially bars and nightclubs, and traditional coffee houses. Improvement is also needed in dining areas in hospitals and other public places, as well as in taxi cabs in some cities.

Laboratory Science:

1st Place - Cailin Deal

Title: Vectored antibody gene delivery protects mice against sporozoite challenge

Plasmodium sporozoites can be neutralized in vitro by monoclonal antibodies (MAb) against the circumsporozoite protein (CSP). Passively transferred MAb against P. falciparum CSP can block liver invasion by sporozoites of a transgenic rodent parasite that expresses P. falciparum CSP (Pb-Pf), preventing infection in mice. Despite this, attempts at targeting CSP for a vaccine have fallen short of expectations, in part due to inability to induce durable high-titer antibodies. A single un-neutralized sporozoite can initiate infection, necessitating sustained high-titer neutralizing antibodies for lasting protection.

Recently, David Baltimore's laboratory developed an adeno-associated virus type 8 (AAV8) platform that efficiently delivers pre-formed MAb genes in vivo and directs sustained, high-level MAb production. With the Baltimore lab, we have adopted that technology to express humanized MAbs against the central repeat region of the CSP protein of P. falciparum in mice. Mice developed high titer human IgG antibodies as early as 1 week post transduction and levels have remained constant for more than 20 weeks at 200 to 1000 µg of IgG/ml. In 70 percent of mice transduced with CSP MAb humanized 2A10 (h2A10), and challenged intravenously with 10,000 Pb-Pf sporozoites, parasite invasion of the liver was reduced to below the level of detection. Up to 70% of h2A10-transduced mice challenged by infected mosquito bite were sterilely protected, and 2A10-transduced mice displayed a statistically significant delay in time to patency.. Examination of antibody levels in individual mice revealed that all mice with human IgG concentrations above 1mg/mL were completely protected from bite challenge. This suggests that exceeding this antibody threshold results in consistent sterile protection and establishes that vectored MAb gene delivery has the potential to be an effective form of malaria control.

2nd Place - Tyna Dao

Title: Environmental Exposure to House Allergens Linked to 5hmC Perturbations in Lung

Background: Asthma is a chronic disease of the airways that affects 235 million people worldwide, making asthma a major public health problem1. Asthma is characterized by lung inflammation and airway narrowing which leads to increased breathlessness and wheezing1. The rising incidence of asthma indicates that genetic predispositions are not the only asthma risk factors. Epigenetic regulation (heritable changes in gene expression that occurs in the absence of alterations in DNA sequences) has been suggested to play an important role in the complex gene-environment interactions that can lead to asthma. Epidemiological evidence associates environmental exposure to tobacco smoke environments, critical risk factors to the development of asthma, to aberrant epigenetic mechanisms2. Epigenetic regulation, including DNA methylation, histone modifications, and non-coding RNA (ncRNA), contributes to gene transcription that affects the function of target cells, tissues and organs. Therefore, epigenetics may provide the link between environmental exposure to pollutants and allergens which triggers alterations in gene expression to biologically alter lung function, resulting in the pathogenesis of asthma.

House dust mite (HDM) exposure is one of the environmental triggers associated with asthma3 but the mechanisms are not well established. HDM is the most prevalent allergen in allergic disease and found commonly in mattresses and pillow covers4. We showed acute5 and chronic6 exposure to HDM increased airway hyperresponsiveness (AHR) through epigenetic modulation of the airway cell phenotypes in mice. AHR is a classical phenotype to diagnose asthma since asthmatic individuals have a lower threshold for methacholine challenge and exhibit increased AHR as a hallmark of asthma or hyper-reactive response to stimuli.

DNA methylation is the best studied epigenetic mechanism, the process that suppresses gene transcription via the addition of a methyl group to the fifth base of cytosine to produce 5-methylcytosine (5mC) 7. However, increasing research has focused on TET (Ten-Eleven Translocation) protein, an epigenetic modulator that catalyzes the oxidation of 5mC into 5-hydroxymethylcytosine (5hmC), which promotes the removal of DNA methylation or blocks DNA methylation machinery7. We found up-regulation of Tet1 and increased 5hmC content in the lung of mice with repeated HDM exposures, possibly indicating a role for Tet1 in the development of AHR.

Objective: This study aims to investigate the role of Tet1 in HDM driven AHR.

Methods: We examined the role of Tet1 in HDM driven AHR in vivo by using a Tet1 mutant murine model (Tet1+/-) sensitized with 100μg HDM (intraperitoneal) and challenged twice with 100 μg HDM (intratracheal) before subjecting to the methacholine challenge for AHR measurement. We measured protein levels of cytokines in the serum by ELISA (Enyzme-linked Immunosorbent Assay) to assess allergic inflammation. We measured the global 5-hmC content in mouse lung and isolated tracheal airway smooth muscle cells (ASMCs) by ELISA. In addition, we translated our mice findings on Tet1 directly to epigenetic changes in human asthmatic ASMCs. We examined the gene expression of TET-1, 2 and 3 and the global 5-hmC

content in human normal and asthmatic ASMCs by real-time PCR and ELISA respectively. TET activity was measured in nuclear extracts by colorimetric assay which detects the TET-converted 5-hmC products. Furthermore, by using small-interfering RNA targeted for TET1 (siTET1) in human asthmatic ASMCs we examined the effect of TET1 knockdown upon gene expression related to airway smooth muscle phenotype (cell proliferation, cell contraction, collagen synthesis).

Results: Tet1+/- mice were protected from HDM-driven AHR compared to wild-type HDM exposed mice. Also, both lung tissues and ASMCs from HDM-exposed Tet1+/- mice showed decreases in HDM-induced 5hmC content compared to wild-type controls. It implies that Tet1 mediates HDM-driven AHR through altering hydroxymethylation patterns in the lung affecting lung function. Additionally, we found human asthmatic ASMCs had increased 5hmC content, TET activity and TET1 mRNA level compared to normal ASMCs suggesting that TET up-regulation is persistent in cultured asthmatic ASMCs. Moreover, we observed the suppression of TET1 in asthmatic ASMCs decreased mRNA levels of genes regulating cell proliferation (PCNA, Proliferating Cell Nuclear Antigen, CCND1, CyclinD1), cell contraction (CAMK2D, Calcium/Calmodulin-Dependent Protein Kinase II Delta) and collagen synthesis (COL3A, Collagen Type3 Alpha),possibly indicating that TET1 plays a role in ASMC phenotype.

Conclusions: Our data suggests that Tet1 plays a distinct role in mediating airway function, possibly through aberrant DNA hydroxymethylation. The results of our studies provide a novel perspective of epigenetic regulation of allergen-driven changes in ASM function and AHR. It addresses the gaps in our understanding of how environmental insults may initiate asthma.

3rd Place - Sivabalan Manivannan

Title: Glutamine antagonist, DON, protects mice from acute fatal encephalomyelitis by inhibiting T-cell growth and proliferation

Inflammation in the nervous system is a necessary part of the response to CNS infection, but also causes neuronal damage in both infectious and autoimmune diseases of the CNS. Sindbis virus is an enveloped, positive-strand RNA virus that causes acute encephalomyelitis and fatal paralysis in mice. Adult C56BL/6 mice inoculated with the neurovirulent strain of Sindbis virus (NSV) succumb to fatal paralysis despite clearing infectious virus resulting from a robust anti-viral adaptive immune response. Pervious studies from our lab have shown that survival after NSV infection is improved in T-cell deficient mice and in mice with pharmacologic inhibition of the inflammatory response indicating that development of treatments that reduce CNS inflammation as well prevent glutamate excitotoxicity as an important therapeutic goal. We show that low daily doses of the non-reversible glutamine antagonist, DON (6-diazo-5-oxo-l-norleucine), rescues mice from fatal paralysis by inhibiting the inflammatory immune response. DON-treated mice fail to induce an adaptive immune response to the virus in the deep cervical lymph nodes and show decreased CD45+ and CD3+ lymphocyte infiltration into the brain and delayed viral clearance compared to vehicle-treated controls. DON-treated mice show significantly reduced mRNA expression of neurotoxic and inflammatory cytokines (TNF-alpha, IL-1b, IL-6, and IFN-gamma) during the course of viral infection in the brain compared to vehicle controls. Additionally, in vitro studies using purified CD3+ T-cells show that DON inhibits the growth and proliferation, but not the activation, of primary mouse CD3+ T-cells in response to stimulation with anti-CD3/CD28. Following stimulation, DON-treated lymphocytes show a defect in S6 phosphorylation, a downstream mTOR pathway target suggesting impaired protein translation in primary lymphocytes. Stimulated DON-treated T-cells have lower IL-2 protein production despite having similar levels of IL-2 mRNA compared to vehicle controls. These studies suggest that small molecules that antagonize glutamine metabolism are immunomodulatory and could be beneficial in the treatment of neuroinflammatory diseases.