Determining Microbial and Neuroimmunological Differences Associated with Adolescent Alcohol Use

Early alcohol use is an important risk factor for alcohol and substance use disorders, but the neurobiological changes that lead to increased vulnerability are not well understood. Alcohol-induced alterations in the microbiome at an early age may lead to neural changes that affect the escalation of alcohol use. Consequently, the microbiome is a novel area of research for adolescent alcohol use disorder (AUD). The present studies investigated differences in the oral microbiome of human adolescents (ages 17-19) who engage in heavy drinking (n=21, 52.4% female) compared to non-drinking control participants (n=18, 44.4% female). We explored associations between the oral microbiome, oral cytokine mRNA levels (interleukin 6 [IL-6], interleukin 1 beta [IL-1β], tumor necrosis factor alpha [TNF-α]), and proton magnetic resonance spectroscopy markers of neuroinflammation (myo-inositol, total choline, total creatine, N-Acetylaspartate). As hypothesized, adolescents who engaged in heavy drinking had significant differences in the diversity and composition of the oral microbiome. Compared to the control group, the alcohol-using group exhibited lower evenness and higher abundances of Rothia and Corynebacterium. We observed no significant differences in cytokine mRNA levels between the groups; however, levels of IL-1β correlated with the abundances of several genera in both groups, including abundances of Rothia. IL-1β was also associated with a higher number of alcohol use days in the past 90 days and phosphatidylethanol concentrations in the alcohol-using group. While there were no significant group differences in neuroinflammation markers, abundances of Rothia were associated with significantly higher concentrations of total creatine-containing metabolites across the whole sample. The associations between Rothia, mRNA cytokine levels, and neurometabolite levels are intriguing, as Rothia is well known for its high capacity to covert alcohol into acetaldehyde, suggesting a potential link between the oral microbiome and neurobiological processes related to alcohol use. Taken together, these studies indicate that the oral microbiome may be affected by alcohol use and is associated with cytokine mRNA levels and neurometabolite concentrations in individuals who use alcohol and controls. The microbiome may aid in prevention and intervention efforts for adolescent AUD by providing new targets for treatment and/or diagnostic and therapeutic response biomarkers

A Comparative Analysis of Births for Women Aged 20-25 Vs. 35-39: An Examination of Longitudinal Trends of Volume and Outcomes

Maternal health impacts the well-being of society as it is commonly viewed as an indicator of public health welfare. Childbirth at an advanced maternal age (AMA) increases the likelihood of less favorable birth outcomes. As births to AMA women have increased over time, it is important to understand the impact age has on birth methods and birth outcomes. This research examined the proportion of births to women aged 20-25 years in comparison to AMA women aged 35-39 years in North Carolina from 2002-2017. The proportions of births to AMA women have increased over time despite increased risk and birth outcomes. An examination of longitudinal trends over 16 years showed more births to women aged 20-25 years, accounting for 73.6% of total births between the 2 age groups. AMA women showed a higher rate of cesarean sections, stillbirths, and longer length of stay following childbirth

Gut Check: Exploring the Role of Acinetobacter in Intestinal Inflammation

Inflammatory Bowel Disease (IBD) is a chronic disorder characterized by excessive intestinal inflammation. Alterations in the gut microbiota have been consistently observed in individuals with IBD, but it is unclear which bacteria participate in driving inflammation. A comprehensive survey of publicly available RNA-sequencing datasets revealed that Acinetobacter species are elevated in the gastrointestinal tract of IBD patients. We found that A. calcoaceticus was particularly elevated in Crohn’s Disease patients; a subset of IBD. It is well documented that Acinetobacter species are resistant to several antibiotics, but there is very little information on the effects of Acinetobacter in the gut and hardly any data on A. calcoaceticus. Computational analysis of Acinetobacter genomes revealed that A. calcoaceticus harbored a wide repertoire of multi-drug efflux pump and beta-lactam resistance genes. We found that A. calcoaceticus strains were moderately to highly resistant to certain antibiotics within fluoroquinolones, aminoglycosides, tetracyclines, and other antibiotic classes. Modeling the conditions of the gut, we found that A. calcoaceticus strains tolerated varying levels of pH, osmolarity, ethanol and hydrogen peroxide. Biolog phenotypic microarrays further revealed that A. calcoaceticuscould use a range of nutrient sources found in the gut, including monosaccharides, disaccharides, glycosides, and amino acids. Moreover, A. calcoaceticus could grow anaerobically in stool-based bioreactors. Using inside-out intestinal organoids, we found that A. calcoaceticus stimulated pro-inflammatory cytokines; suggesting that A. calcoaceticus could initiate inflammation. In vivo, we found that A. calcoaceticus did not stimulate inflammation in the setting of a complex gut microbiota. However, disruption of the gut microbiota with antibiotics resulted in enhanced inflammation in A. calcoaceticus treated mice. To model IBD, we administered 2,4,6- trinitrobenzene sulfonic acid (TNBS) and we observed that TNBS treated mice receiving A. calcoaceticus lost more weight and had worse histological scores than mice treated with vehicle control and TNBS; indicating Acinetobacter worsens intestinal inflammation. Addition of antibiotics to the TNBS model further heightened the inflammation induced by A. calcoaceticus. Collectively, these data demonstrate that A. calcoaceticus is antibiotic resistant, well adapted to survive the gastrointestinal tract, and is capable of initiating inflammation in the setting of an altered gut microbiota

Next Generation Human Cardiac Organoids: Modeling Inflammatory Diseases and Engineering Their Protection in vivo

In the context of excessive inflammation, cardiovascular complications such as arrhythmias, functional deficits, vascular dysfunction and fibrosis have all been reported. However, characterizing these pathologies has remained challenging given the complex interplay of cytokines, immune cells, and the affected tissue. Given the immune system’s known roles in remodeling post ischemic injury, preclinical efforts have been directed towards minimizing excessive fibrosis and immune cell infiltration. To facilitate the healing process and spare the myocardium from pathological remodeling and functional decline, cellular transplantation therapies have been employed, with the intent to either replace damaged cardiomyocytes, or provide a continued source of healing factors to the affected area. However, the inability to produce universally transplantable organoids capable of avoiding immune rejection has limited advances in engineering approaches aimed at regenerating damaged human myocardium. Animal models and 2D cultures of human cells have been employed to study the effects of cytokines, immune cells, and infectious insults on the heart, but their translational impact has been limited due to interspecies differences, cell-cell communication, and the significance of three-dimensional tissue organization. To this end, our lab has developed human cardiac organoids for disease modeling and regenerative medicine. Given their ability to recapitulate key hallmarks of myocardial infarctions and drug induced cardiotoxicity and the intrinsic inflammatory properties of the cell types composing the organoids, we hypothesized these human cardiac organoids would provide a suitable platform to model myocardial inflammation. We further hypothesized the vasculature in our cardiac organoids could be leveraged to engineer a vascularized cardiac microtissue. In doing so, the vascular network would address current limitations such as nutrient accessibility and enable the incorporation of immune cells. Finally, we hypothesized that engineering T cells, or the organoid graft to abrogate the host immune response would prolong organoid survival time to increase their therapeutic benefit. In the enclosed dissertation, we illustrate these human cardiac organoids have the potential to model inflammatory conditions such as the acute cardiac injuries induced by COVID-19 Cytokine Storm, to be engineered into a perfusable microtissue, and show that multiple immune engineering approaches have the potential to augment cellular transplantation therapies in the context of cardiac regeneration post myocardial infarction

MALDI-MSI Identification of Tissue-Level N-Glycomic and Proteomic Molecular Biomarkers of Aggressive Prostate Cancer

Prostate Cancer (PCa) poses a significant clinical challenge, characterized by debates on screening efficacy and continuous issues with distinguishing aggressive from non-aggressive tumors. Despite treatment advancements, poor patient outcome at late stages and clinical overtreatment of indolent disease emphasizes the urgent need for markers of PCa aggression. Aberrant glycosylation and extracellular matrix (ECM) remodeling are two complex molecular processes necessary for PCa progression, highlighting a novel area for biomarker discovery. Leveraging formalin-fixed paraffin-embedded tissues, tissue microarrays (TMAs), and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), distinct N-glycan and ECM profiles are identified. Elevated bisecting, multi-antennary, and fucosylated N-glycans indicate early signs of increased risk for PCa metastasis in a novel patient outcome TMA cohort. Serial enzymatic digests of tissues revealed samples rich in N-glycans with core-fucose modifications, specifically, exhibited greater risk for eventual metastasis. Further analysis of large tissue samples from which TMAs were derived revealed a unique spatial distribution of these N-glycans of interest throughout both tumor and tumor-adjacent microenvironments, providing novel vi insight into the roles of these structures in disease progression. In further pursuit of increased fucosylation as a marker for PCa aggression, analysis of a TMA constructed from tissues with heightened risk of neuroendocrine differentiation and castrate-resistance revealed a greater abundance of fucosylated N-glycans. Investigating differences in ECM composition between tissue samples from PCa that eventually metastasized and PCa that never recurred sheds light on potential prognostic markers that have potential to improve patient outcome. Proteomic analysis of these tissues revealed several protein species exclusive to PCa at risk for metastasis. These biomarkers hold transformative implications for tailoring patient-centric treatment strategies and foster a comprehensive understanding of disease dynamics at a molecular level

Tier One Tools: Improving Educators’ Understanding of the Multi-Tiered System of Supports Within the School System Including a Case Example

South Carolina public school teachers provide intervention services via 2 models: Response to Intervention (RtI) and the Multi-Tiered Support System (MTSS). Research shows that there is currently a gap in teacher understanding and implementation of MTSS versus RtI within South Carolina public school districts. Teachers noted lack of training, time, and resources as the major barriers to implementation and would benefit from two sets of modules: 1) an educational training module on MTSS and 2) example intervention modules that can be implemented within their classrooms at a tier one level in K-5 classrooms within the public school system in Florence School District 1. Data collected at the end of the 14-week capstone experience showed that teachers had an improved understanding of MTSS referrals when utilizing the training information provided. Teachers also showed confidence in implementing Tier One interventions in their classrooms to meet the needs of their students