Septic cardiomyopathy is a significant heart muscle dysfunction that often occurs during life threatening systemic infection (sepsis) and correlates with higher mortality. Currently, it is unknown why some septic patients develop such cardiomyopathy, while others do not. When used as models for inflammatory responses in human patients, rodents have historically been poor predictors of clinical outcome. For the current study on septic cardiomyopathy, I have therefore created heart cells (cardiomyocytes) from human stem cells. These were derived from two groups of septic patients: one with and one without septic cardiomyopathy, representing experimental and control groups, respectively. After maturing these cardiomyocytes, we treated them with specific inflammatory signaling molecules (TNF alpha, IL1b, IL6) and recorded video footage of the cells’ contraction response. The video recordings were then processed by a Matlab script that tracks the cardiomyocytes’ contraction and calculates their contractile velocity and amplitude under each condition. In response to these specific inflammatory mediators, we anticipate a significant change in contractility in cells from septic patients with cardiomyopathy, compared to the control cells from septic patients without cardiomyopathy. If our assumption is correct, further comparison of these cells will allow us to identify molecular signals that trigger septic cardiomyopathy. Such signaling pathways could then be further targeted as novel therapeutic strategies.

The female reproductive tract must maintain normal reproductive functions while also being able to elicit immune responses to sexually transmitted microbes and viral pathogens. In response to virus exposure, the balance of these two functions can determine the outcome of infection and disease through alteration of vaginal mucosa integrity. Recent in vivo research in our laboratory revealed that vaginal infection by Zika virus (ZIKV), an emerging mosquito-transmitted flavivirus that is also sexually transmitted among humans, induces epithelial cell-specific innate immune response that accelerates a homeostatic form of cell death known as cornification. We hypothesize that specific processes in vaginal epithelial cells mediate cornification in response to ZIKV infection, and that this cornification may alter barrier properties, innate-adaptive immune crosstalk, and ultimately, the degree to which ZIKV disseminates from the female reproductive tract. We therefore analyzed and compared vaginal epithelial cell infection by both African and Asian lineage ZIKV strains using traditional 2-dimensional cell culture and organotypic 3-dimensional culture infection models. Differences in viral infection and replication kinetics, and innate immune response were characterized through RT-qPCR, immunoblot analysis, cell imaging, and viral plaque assay. Ongoing analyses are expected to reveal the application of organotypic 3-dimensional cultures in capturing in vivo qualities of vaginal epithelial cell/ZIKV infection and response compared to 2-dimension cultures. These studies will provide insights for application of vaginal epithelial cell culture models of ZIKV infection that encapsulates the complex functional and structural aspects present in vivo.

Porphyromonas gingivalis is a bacterium that is often present in the gum pockets of people who have gum disease. Research has indicated that P. gingivalis is able to avoid detection by a host’s immune system by modifying the lipid A structure on its outer membrane. Our research has examined the effect of lipid A modifications on outer membrane vesicle production. Outer membrane vesicles (OMVs) are formed when the outer membrane of the bacterium bends out into a tight bubble-like shape and pinches off from the membrane completely. As this happens, contents within the membrane space can be packaged inside of the vesicle. OMVs may contain toxins or misfolded proteins for expulsion from the bacterium, or they can package DNA which enables horizontal gene transfer. It is known that OMVs are common in Gram-negative bacteria, but the exact mechanism of how they are formed is not yet well understood. To better understand OMV production, I have performed assays to quantify the OMV production of several different strains of P. gingivalis. Mutant strains provided to our lab had their lipid A protein mutated as follows: dephosphorylated and tetra-acylated (wild type), phosphorylated at the C4 position (Δ1587), phosphorylated at the C1 position (Δ1773), and penta-acylated (Δ1123). Assay results indicated that when compared to wild type, phosphorylation of the C4 position (present in Δ1587) inhibits OMV production, and yet phosphorylation of the C1 position (present in Δ1773) enhances OMV production. Whether lipid A has 4 or 5 acyl chains (Δ1123), the difference appears to have no significant effect on OMV production. The logical next step is to identify which proteins are causing these differences by their varying interactions with lipid A. Identifying these proteins will contribute to the larger scientific quest of understanding how outer membrane vesicles are formed.

Periodontal disease stands to be the leading cause of tooth loss in older adults and is characterized by inflammation of tissues supporting the teeth and periodontal bone loss. This oral disease also occurs during normative aging in mice models. Thus, targeting the biological aging process may provide an innovative approach to reduce the impact of this disease in the elderly population. Rapamycin is a specific mTOR inhibitor shown to delay age-related decline and significantly extend the lifespan of mice models. Our lab has demonstrated that an 8 week treatment with rapamycin in aged animals reversed age-related periodontal disease. However, whether this improvement persists even after the treatment ends is still unknown. To determine whether reversal of periodontal disease persists even after stopping rapamycin treatment, cohorts of aged mice were either started with 8 week treatment with control (eudragit) food or rapamycin (42ppm) food, and then switched to either rapamycin (42ppm) food or control (eudragit) food for another 8 weeks, respectively. High resolution microCT imaging was completed to measure the amount of periodontal bone, and western blot was performed on total alveolar bone extracts and probed for the osteoclast marker RANKL. Our microCT analysis showed there was less periodontal bone loss in aged mice treated with rapamycin, and this result persisted even after 8 weeks on the control diet. Additionally, the age-related increase in RANKL expression was decreased in both treatment groups. In conclusion, the regain of lost periodontal bone in aged mice persisted even after cessation of rapamycin treatment, and the age-related increase in the osteoclast marker RANKL was decreased after rapamycin treatment and such decrease persisted. Overall, our studies indicate that short term treatment with rapamycin is sufficient to rejuvenate oral health and the benefits are maintained even after stopping the treatment.

The focus of this project is to study two newly discovered gene fusions that involve the Anaplastic Tyrosine Kinase (ALK) gene and define their role in Acute Myeloid Leukemia (AML) and potential for utility as therapeutic targets. ALK is a Receptor Tyrosine Kinase that contributes to brain development, regulation of cellular functions, and is linked to cell growth. Mutations and other genetic alterations are identified in various cancers. We recently discovered novel ALK fusions within a small set of AML patients in individuals with Monosomy7 AML (Loss of chromosome 7) and one of the most refractory subtypes of AML, making this particular group of patients high-risk. Additionally, ALK is otherwise not expressed in normal bone marrow making this a potential target. The current focus has been to verify if the ALK fusions are viable gene targets for chemotherapies that are already approved for clinical use, specifically Crizotinib, a chemotherapy drug and Tyrosine Kinase Inhibitor; by focusing on an FDA-approved targeted chemotherapy drug, this eliminates much of the process and time it would take to be able to treat AML in these high-risk patients. Crizotinib is successful in treating abnormal ALK expression seen in Anaplastic Lymphoma, Neuroblastoma, and Non-Small Cell Lung Cancer (NSCLC), and functions by preventing critical cell signals. We have engineered AML cell lines to express these fusions of interest in order to test Crizotinib in the context of AML.

Bile acids are intestinal metabolites that are biotransformed into diverse secondary bile acids to aid with digestion and absorption. However, once modified by the gut microbiome, they can produce serious health implications including colorectal cancer risk. We hypothesized that this bile acid metabolism is reflected in bacterial cell morphologic changes. To test this hypothesis, we anaerobically cultured and generated light microscopy images of Clostridium scindens in media containing 100 μM cholic acid (a known substrate in the production of the carcinogenic bile acid deoxycholic acid), C. scindens in media containing NaCl (a positive control; 1% NaCl is shown to cause shrinkage through osmosis in bacterial cells), and C. scindens in media alone (negative control) (8 images per group; 500 bacterial cells per image). We developed an image-based model using MicrobeJ (an ImageJ plug-in developed for analysis of bacterial images) by using smoothed particle contours and a skeletonization algorithm adjusting parameters to accurately detect cells. We observed a significant difference in shape descriptor analysis between curvature of the end points and center of the medial axes, width of the medial axes, ratio between the major and minor axes of the cells, ratio between area and convex area, angularity, roundness, length of the medial axes, circularity, and perimeter of the outside boundary between C. scindens with and without the presence of cholic acid (p < 10^-5 for all comparisons; Welch’s two-tailed t-test). Of note, this represented a larger difference than the delta between the two controls. Our data demonstrate that image-based analysis can enable detection of cellular morphologic differences of C. scindens based on metabolic profile with respect to bile acids. In principle, this approach could be expanded to other bile acids and/or beyond bile acid metabolism to identify bacterial metabolic behaviors of interest (e.g. assessing or predicting effects of clinically relevant compounds targeting the microbiome).

 Cancer recurrence and migration remains a persisting challenge in oncology. As such, accurate models of tumor growth in the laboratory (in vitro) are critical to our continued understanding and treatment of the disease. This project aims to use one such model—the 3-dimensional spheroid—to measure the effects of novel targeted therapy combinations on different cell lines. Two cell lines were explored in this experiment: UM-SCC74A (a tongue squamous cell carcinoma) and HCT116 (a colorectal carcinoma). Spheroids were formed by culturing cells in ultra-low attachment well plates for 24 hours, allowing the cells to coalesce and form a 3-dimensional sphere. An extracellular matrix was added in some trials to further improve the accuracy of the model. Spheroids were cultured in the presence of drug for 48 hours, then allowed to grow in drug-free media following washout. Spheroid size and viability were assessed via imaging and Cell-Titer Glo assays, respectively. The results from both cell lines echoed those obtained in 2-dimensional assays and validated the efficacy of our drug combinations in a 3-dimensional setting. These models could provide an intermediate step between 2-dimensional in vitro experiments and those conducted in live organisms (in vivo). The success of this model in supporting different cell lines affirms the robustness of this approach and its potential for use with a wide variety of cancers.