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Delta Omega

2015 Poster Competition Winners

Laboratory Science

1st Place - Cissy Li (Overall Winner) (PhD student)

Title: Candida albicans superoxide dismutases as part of an adaptive response to copper during infection 

 

Candida albicans is the most prevalent human fungal pathogen.  The organism usually exists as a harmless commensal, its growth held in check by the host immune system and the surrounding microflora.  However, when the opportunity arises, it can become infectious and even fatal by targeting numerous organ systems.  Immunocompromised individuals such as HIV, surgery, chemotherapy patients, and premature infants are all susceptible to these lethal infections.  Although antifungal drugs can help control the occurrence and severity or infection, incidence is still on the rise, especially in hospital settings.  Thus C. albicans infection remains a public health threat.

To thrive in the many diverse niches of a mammalian host, C. albicans must be highly adaptive.  One challenge faced is how to sustain metal homeostasis when subjected to extremes in host metal availability.  Like all organisms, the human fungal pathogen C. albicans needs to carefully control its acquisition of heavy metals such as copper.  Since copper is both an essential nutrient and a potential toxin, copper availability at the host-pathogen interface can be key for pathogen fitness and survival.  During infection, host copper bioavailability can increase dramatically in an attempt to thwart pathogen growth through copper toxicity.  Stimulated macrophages can expel extraordinarily high levels of copper into the phagolysosome, which is paired with a systemic increase in serum copper bioavailability. 

Copper is not only toxic, it is an essential nutrient for fungal pathogens.  In C. albicans, copper is required for many essential pathways, including as a co-factor for superoxide dismutase (SOD) enzymes.  SODs play important roles in oxidative stress defense, and curiously C. albicans has evolved with an unusually large family of SODs.  While most eukaryotes express only two or three SODs, C. albicans has six.  One is a highly irregular manganese-containing Sod3 that resides in the same cytosolic compartment as a Cu/Zn-Sod1.  The vast majority of eukaryotes have only a Cu/Zn-SOD in the cytosol, and a few rare organisms (crustaceans and photosynthetic microbes) contain a cytosolic Mn-SOD but no Cu/Zn-SOD.  C. albicans and related fungi are the only organisms known to have both.  The rationale for this apparent redundancy in SODs with different metal co-factors is the focus of our research.

In Dr. Valeria Culotta’s lab, we investigated the basis for overlapping SODs in the C. albicans cytosol and have uncovered a new adaptation to copper deficiency in this yeast.  We observe that laboratory cultures of C. albicans with abundant copper exclusively express Cu/Zn-Sod1.  But as cultures are depleted of copper, cells will switch to expressing Mn-Sod3 using a novel transcriptional control mechanism involving the copper-sensing transcription factor Mac1.  This shift from Cu/Zn-Sod1 to Mn-Sod3 was observed in several clinical isolates of C. albicans, and most importantly, in a murine model of disseminated candidiasis.  During the course of kidney infection, kidney copper levels dropped.  Meanwhile, C. albicans induced the shift from Cu/Zn-Sod1 to Mn-Sod3 to adapt to this copper deficient environment.  Our results show that C. albicans is well equipped to adapt to variations in copper availability of the animal host.  Furthermore, this is the first evidence that the host may use copper deprivation as a strategy to fight microbial infection.  These new findings on C. albicans pathogenesis may lead to the identification of novel and specific antifungal drug targets.


2nd Place - Justin Jacob (PhD student)

Title: Novel nuclear functions for a keratin intermediate filament protein in skin

Principal Investigator: Dr. Pierre A. Coulombe Department: Biochemistry and Molecular Biology

Keratins, the largest subfamily of intermediate filament (IF) proteins, are predominantly expressed in cells of epithelial origin where they are tightly regulated in a cell type-, tissue- and context-dependent manner1. There are 28 Type I (acidic) and 26 Type II (neutral/basic)1,2 keratins that form obligatory heteropolymers as they self-assemble into 10 nm filament networks in the cytoplasm of epithelial cells. Keratin IFs play major roles in mechanical support, cytoarchitecture and, of recent interest, signal modulation during cell growth, migration, and apoptosis1,3, with implications for human diseases such as cancer. Since greater than 80% of adult tumors are epithelial cell-derived4, the signaling roles of keratins may have significant implications in cancer progression and metastasis.

Keratin 17 (K17), a 46-kilodalton type I keratin protein, is positively correlated with epithelial carcinomas such as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC)5-7. K17 is constitutively expressed in epidermal appendages such as hair follicles, nails, and glands, and is inducibly expressed in activated keratinocytes in response to skin injury and other challenges8-10 and in diseases such as psoriasis11 and cancer5-7. Within the past decade, several studies have identified a novel role of K17 in signal modulation in both proliferative and pro-inflammatory contexts5,8,10. Furthermore, interactions between K17 and nuclear-translocating proteins such as Importin-13, Annexin A2, AIRE, hnRNP K and HSP70 (refs. 10,12,13; also, unpublished observations from our lab), as well as its ability to positively regulate the expression of several immune response genes (such as Aire and pro-inflammatory cytokines Mmp9, Cxcl10, Cxcl11, Ccl19, IL-4, and IL-6) (R. Hobbs et al.14, manuscript submitted), have recently come to the fore in our laboratory, though the associated significance is only partly understood.

Given evidence suggesting that the co-localization of HSP70 and AIRE within the nucleus is K17-dependent14, the previously unspoken question now emerges: Could K17, a cytoplasmic intermediate filament protein, modulate gene expression by acting inside the cell nucleus? The widely accepted notion is that, with the notable exception of the nuclear lamins, all IF proteins are confined to the cytoplasm15 . Similar biases were once held for actin and tubulin, which have now been essentially refuted in light of extensive data revealing their presence and function within cell nuclei16,17.

The current project focuses on defining the mechanisms involved in K17-mediated signaling and, in particular, on K17’s presence and function within the nucleus of tumor-prone, or inflammation-prone, keratinocytes. When A431 (human epidermoid carcinoma), HeLa (human cervical adenocarcinoma), and mouse epidermal keratinocyte (MEK) cells are treated with Leptomycin B (LMB, an inhibitor of Exportin-1) to selectively interfere with nuclear export18, we find that K17, along with its type II keratin partner K5, occurs as distinct puncta within the nucleoplasm when visualized by confocal laser scanning microscopy14. Ongoing efforts in the Coulombe laboratory have already revealed a similar punctate pattern for the proteins AIRE and HSP70, which as mentioned above colocalize in the nucleus in a K17-dependent fashion. Both AIRE and HSP70 are K17-binding proteins13 (R. Hobbs and B.M. Chung, unpublished observations), and we have evidence that the K17-AIRE interaction is instrumental in modulating expression of several pro-inflammatory genes in tumor-prone human and mouse skin keratinocytes. Experiments utilizing Exportin-1- inhibited A431 cells transfected with mCherry-Aire have so far shown that K17 puncta co- localize with those of mCherry-AIRE in the nucleus.

Finally, nuclear K17 can be visualized in tissue sections of patient biopsies containing basal cell carcinomas, revealing a potential physiological relevance for K17 nuclear localization in human disease such as cancer 14. In an effort to investigate novel functions of nuclear K17, chromatin immunoprecipitation (ChIP) assays were performed in A431 cells treated with the phorbol ester, TPA, to induce a characteristic pro-inflammatory transcriptional response. Data obtained from these experiments reveal a robust physical association of K17 with specific sequences in the 5’-upstream promoter regions of relevant pro-inflammatory and immune response genes (i.e. Mmp9, Cxcl10, Cxcl11, and Ccl19) 14, suggesting its involvement in steps related to transcriptional regulation. Interestingly, the segments of DNA that interact with K17 contain consensus binding sequences for the transcription factor p65/NF-κB, which has been demonstrated to play an essential role in skin inflammation and carcinogenesis19. This p65 consensus binding sequence was later used to generate a 32P-radiolabeled DNA probe for use in electrophoretic mobility shift assays (EMSAs).

The results obtained provide additional evidence of a specific association between the probe and a K17- and p65-positive protein complex in nuclear lysates extracted from TPA-stimulated A431 cells14. The functional significance of these physical interactions is currently being investigated through a variety of molecular assays. In conclusion, these findings point to novel roles of K17, a keratin intermediate filament protein, within the nucleus of tumor-prone keratinocytes, opening the field to new lines of inquiry that can have profound implications in signal modulation, gene expression, inflammation, and cancer. In addition, they may provide significant clues regarding the mechanistic basis for K17’s ability to promote the growth and/or aggressiveness of several types of epithelial-based tumors.


3rd Place - Andrea Hodgson (PhD student)

Title: Selective regulation of NF-KB by cleavage of p65 and interfering with the p65/RPS3 interaction by A/E pathogen

The nuclear factor kappaB (NF-κB) signaling pathway has long been recognized as a crucial activator and regulator of the innate and adaptive immune responses. More recently, ribosomal protein S3 (RPS3) has been identified as a non-rel component of NF-κB and functions as a “specifier” protein critical for the induction of selective proinflammatory gene transcription. Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases, which remain an immense health and economic burden worldwide. Colonizing the host gastrointestinal tract, the A/E bacteria inject an array of virulence proteins into colon epithelial cells (CECs) through the type III secretion system (T3SS). A/E pathogens have evolved many strategies to evade the host immune response and encode various effector proteins that target NF-κB at different stages. Our work here, demonstrates how the A/E encoded metalloprotease NleC, specifically cleaves the p65 molecule at the N-terminus generating a fragment that is capable of selectively blocking the function of RPS3. This selective blockade of RPS3 results in a dampened immune response following infection, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases.

 

Applied/Epidemiology

1st Place - Dustin Gibson (PhD Student)

Title: The Mobile Solutions for Immunization (M-SIMU) Trial: A preliminary analysis for a cluster randomized controlled trial that assesses the impact of mobile phone delivered reminders and incentives to improve timely childhood immunization in western Kenya

Introduction: Short message system (SMS) reminders and incentives are two demand side interventions that have been shown to improve healthcare seeking behaviors in lower income countries.  We sought to determine if these interventions could improve timeliness of pediatric immunizations in rural western Kenya.

Discussion: Infants of caregivers who received SMS reminders + 200 KSH incentives were significantly more likely to receive pentavalent3 vaccination.  Monetary incentives and mobile phone technologies may improve timely immunization coverage estimates in resource-constrained settings.

 

2nd Place - Anna Fretz (MHS Student)

Title: The Association of Socioeconomic Status with Subclinical Myocardial Damage

Background: The association between socioeconomic status (SES) and clinical cardiovascular events is well established. However, little is known about the relationship between SES and subclinical myocardial damage, as assessed by a novel highly sensitive assay for cardiac troponin T (hs-cTnT).

Conclusions: Low SES was associated with elevated hs-cTnT cross-sectionally, independent of cardiovascular risk factors. These results were stronger in blacks, both cross-sectionally and prospectively. Given the strong association between elevated hs-cTnT and risk of incident cardiovascular events, those with low socioeconomic status, particularly blacks, should be targeted aggressively for cardiovascular risk reduction. Further research is also needed to explore how low SES contributes to subclinical myocardial damage. 

 

3rd Place - Sara Rasmussen (PhD Student)

Title: Marcellus Shale Development, Air Pollution, and Loss of Asthma Control

Since 2005, unconventional natural gas development (UNGD) has rapidly grown in Pennsylvania. Each stage of well development has air pollution impacts, but there are no prior epidemiologic studies of UNGD and asthma outcomes. This study used a nested case-control design to evaluate associations between exposure to UNGD and asthma exacerbations in a cohort of Geisinger patients. We evaluated three types of asthma exacerbations: oral corticosteroid medication orders, asthma emergency department visits, and asthma hospitalizations; termed mild, moderate, and severe exacerbations respectively. Our preliminary models found that there is a consistent association between the 4th quartile of exposure of each UNGD phase and each type of exacerbation. 

 

 

Policy/Practice

1st Place - Tatyana Lyapustina (MPH Student)

Title: Impact of Texas's 2010 'Pill Mill' Law on Opioid Prescribing and Utilization

One of the ways that states have addressed the misuse and abuse of prescription opioid pain relievers (OPRs) is through strengthening the regulation of pain management clinics; however, the effect of such measures remains unclear. In 2009, Texas passed legislation requiring all pain management clinics to be certified by the Texas Medical Board and to be owned and operated by a licensed physician, effective September 1, 2010. We quantified the effect of this legislation on OPR prescription and utilization using IMS Health’s LifeLink LRx data, which represent anonymized, longitudinal, patient-level retail prescription claims capturing approximately 65% of retail transactions in the United States. We applied a segmented regression analysis to this data, focusing on a 24-month observation period spanning from September 1, 2009 through August 31, 2011. We limited our analysis to a closed cohort of individuals with claims activity at the beginning and end of the study period. Our primary outcome measures included average morphine equivalent daily dose (MEDD) per transaction, average monthly MEDD per transaction, and total monthly OPR volume. We modeled the effect of the legislation as a comparison between post-intervention primary outcome values and the counterfactual value, which was estimated for corresponding time points based on pre-intervention levels and trends.  We conducted analyses across all patients and prescribers, as well as stratified by baseline OPR utilization and prescribing. Sensitivity analyses were conducted by varying the inclusion criteria for the closed cohort and by varying the observation period to 18 and 6 months before and after the legislation. Data analysis is ongoing.  The results of this investigation will contribute to an understanding of the extent to which the regulation of pain management clinics, as was done in Texas, impacts OPR prescription and utilization.

 

2nd Place - Elizabeth Harvey (PhD student)

Title: A Community-Based Investigation into Low Birth Weight Deliveries and Collaboration to Improve Maternal and Infant Outcomes at a Baltimore City Hospital

Purpose: 1) To examine characteristics associated with low birth weight (LBW) deliveries at a city community hospital, Mercy Medical Center (MMC), which serves a vulnerable population; 2) To engage in data translation and strategic collaborations with obstetricians and community services to improve birth outcomes.

Conclusions: Both maternal pre-existing conditions and those occurring during pregnancy were found to be associated with LBW. In particular, the increased odds of LBW among women with hypertension before and during pregnancy represents a need to focus on maternal chronic conditions at the community level. Based on community collaborations, one strategy to address pregnant women at risk of LBW infants is to improve the intake and referral system, HCAM, to increase the proportion of PRA forms submitted by practices, increase medical providers rather than administrative staff completing PRA forms, and educate providers of need to resubmit a PRA if a woman’s condition changes. The ultimate objective is better triage of women with increased risk of LBW to appropriate services in the community. Indeed, other research shows similar risk of LBW with maternal complications; these complications account for a significant number of infant deaths as well. Education sessions with providers about PRA forms are one ongoing strategy to improve birth outcomes in Baltimore City.

 

3rd Place - Marta Wilson-Barthes (MSPH student)

Title: Estimating the potential demand and economic impact of a novel tetravalent  dengue vaccine in Brazil

Background and Objectives: Like many countries, the decision to introduce a novel vaccine in Brazil relies largely upon factors related to product manufacturing capacity, supply constraints, potential product demand, and financial considerations. Understanding these issues includes accounting for the various stakeholders involved in vaccine introduction and aligning their priorities within strategic and financial planning frameworks.  The study objectives were threefold: (i) to generate new evidence on the potential demand of the one-dose Butantan tetravalent vaccine for dengue fever in Brazil; (ii) to estimate the costs associated with this demand and (iii) to estimate the disease impact of this demand.

Conclusion:  The decision to accelerate vaccine introduction will depend on the age of the cohort, supply constraints, vaccine price, and timing of licensure. Ongoing analysis is projecting vaccine impact through treatment costs averted while considering staggered state-level introduction based on capacity ranking.