Vibrio cholerae is a natural inhabitant of aquatic environments and serves as the etiological agent for the severe diarrheal disease, cholera. Cholera epidemics follow a regular seasonal pattern, which account for tens to hundreds of thousands of deaths in a given year. V. choleraenaturally persist between epidemics through entry into a dormant state known as viable but nonculturable (VBNC). Research has shown that V. choleraein this VBNC state experience drastic morphological and metabolic changes, which serve as survival mechanisms until environmental conditions become suitable again. The natural marine ecosystem that V. cholerae inhabitis comprised of a complex combination of biotic and abiotic factors, which influence their growth and survival. Some of these factors include interactions with other marine dwellers and environmental pressures, such as fluctuations in temperature and oxygen concentration. Of great interest, we have currently elucidated the role of oxygen on the classical O395 biotype of V. cholerae. Through this discovery, we are especially interested in the role of oxygen and biotype on V. cholerae persistence and how these variables can influence entry into the VBNC dormant state. Specifically, in this study we will compare culturability between the O395 and N16961 biotypes in both aeration and static (non-aeration environments) at 4°C and 30°C. It is of crucial importance to elucidate the role of abiotic environmental factors involved with entry into this dormant state and to understand how V. cholerae have evolved their genetic mechanisms to persist in their natural environments through comparison of biotypes. Understanding entry into the VBNC state is significant because V. cholerae found in this dormant state remain a threat to the human population due their ability to resuscitate and infect the human host under the appropriate environmental conditions.

Plasmodium falciparum, the primary causative agent of malaria in humans, is responsible for life-threatening infections and disease in many tropical regions of the world. In 2018, there were over 228 million cases and 405,000 deaths due to malaria infection, according to World Health Organization estimates. While there has been recent progress in decreasing mortality rates attributed to malaria, the emergence of widespread antimalarial drug resistance in recent decades has endangered such progress. Therefore, there is an urgent need for new antimalarial drugs with a novel mechanism of action. Plasmodium kinases could serve as attractive drug targets due to their essential functions in all stages of the parasite’s life cycle. Plasmodium falciparum Aurora-related kinases (PfArks) have essential regulatory roles in all stages of the parasite’s asexual intraerythrocytic life cycle. As a result, it is hypothesized that PfArks are excellent potential molecular targets for novel antimalarial development. The intent of this study was to identify potent and selective inhibitors of Plasmodium from an Aurora kinase-focused commercial inhibitor library of 3,000 compounds. An initial phenotypic screen was performed at a fixed inhibitor concentration of one micromolar to identify novel compounds with potency against the P. falciparum chloroquine-resistant Dd2 strain. From this library, we have identified multiple compounds with submicromolar antiplasmodial activity in asexual intraerythrocytic life cycle stages and adequate selectivity. Additionally, this project aimed further to characterize the cellular mechanism of action of hit compounds. Multiple compounds were found to exhibit inhibitory effects against early intraerythrocytic asexual life cycle stages as well as liver stages. At this time, one hit compound (DC-6275) was found to inhibit asexual intraerythrocytic as well as liver stage parasites in addition to PfArk1 amongst other Plasmodium protein kinases tested. Overall, we believe that these identified compounds have great potential to serve as scaffolds for future antimalarial drug development.

ABSTRACT It has been widely recognized that microRNAs are involved in nearly all cellular processes that have been investigated and contribute to a variety of diseases including cancer. Our prior studies demonstrated the depletion of one-carbon metabolism related B-vitamins, including folate, vitamin B2, B6 and B12, induced a genomic DNA hypomethylation and an elevation of the tumorigenic Wnt signaling in mouse colonic epithelium. The present study aimed to define whether microRNAs serve as mediators between these B-vitamins and the Wnt signaling, and thereby influence intestinal tumorigenesis. MicroRNA expression profiles were measured using miRNA microarray and real-time PCR on colonic epithelial cells from Apc1638N mice fed with diets deplete or sufficient in those B-vitamins. In silico bioinformatic analysis were performed to predict microRNA gene targets within the Wnt signaling cascade. Out of 609 microRNA examined, 18 microRNAs were found to be either significantly (p < 0.05) or mildly (p < 0.10) differentially expressed in the colonic epithelium of mice fed the depleted diet when compared to the counterpart. Bioinformatic prediction of microRNA gene targets identified 40 genes within the Wnt pathway to have homology with microRNA seed sequences within their 3’-UTR or protein coding sequence. Of the 6 genes tested for experimentally target validation, the expression of Sfrp1 was shown to be significantly inhibited (p < 0.05) whereas β-catenin was shown to be significantly elevated (p < 0.05) with alterations of others in a fashion indicating the activation of Wnt signaling. These findings indicate that microRNAs may constitute a mechanism by which one-carbon B-vitamin depletions regulate the Wnt signaling pathway and thereby inform intestinal tumorigenesis.

Uncontrolled growth of vascular stem cells as a result of endothelial-mesenchymal transition is considered to cause hyper-proliferative vascular remodeling in severe pulmonary arterial hypertension (PAH) patients. Hyperplastic intimal growth is one of the causes of closure of the lumen of pulmonary arterioles. This abnormal vessel remodeling leads to the progressive increase in pressure of the pulmonary arterioles causing severe PAH; and debilitating harm to patients resulting in mortality from right heart failure. Environmental factors, including polychlorinated biphenyls (PCBs), are considered to be involved in hyper-proliferative vascular remodeling because genetic makeup can only explain about 10% of severe PAH cases. PCB involvement in lung toxicity has received attention because (i) they have been reported to accumulate in the lung; (ii) PCBs produce pathological vascular remodeling in the experimental model; high levels of PCBs are found in human lung tissue; and (iii) epidemiological studies show the association between lung toxicity and PCBs; and prevalence of hypertension and elevated concentrations of particularly PCB153. Recent studies identify PCB153 as one of the largest contributors for total PCB body burden in humans. Our previous studies demonstrated PCB153 mediated vascular endothelial dysfunction and activated the inhibitor of differentiation protein 3 (ID3). ID3 is an important determinant of mitogen and reactive oxygen species-induced G1→S phase cell cycle progression. Although phosphorylation of ID3 increases cell growth by antagonizing the transcription of cell cycle inhibitors, still there is a critical gap in understanding the molecular mechanism(s) of pulmonary proliferative vascular remodeling associated with PCB exposure in humans and the role of the transcription regulator ID3. Our overall objective was to investigate ID3 mediated transcriptional reprogramming as a driver of PCB153-induced pathological proliferative vascular remodeling. Stable ectopic expression of ID3 in lung endothelial cells contributed to endothelial-mesenchymal transition (EndMT), cell proliferation, and cell migration. Using an endothelial spheroid assay, an established method to measure aberrant hyper-proliferation of endothelial cells in PAH patients, we show that stable ectopic expression of ID3 increased the number and size of vascular spheres. ID3 overexpressing cells exposed to environmentally relevant concentrations of PCB153 showed a two-fold increase in cell proliferation as determined by MTT, SRB, and BrdU assays. ID3 overexpressing cells showed the loss of VE-cadherin and gain of MMP9 and vimentin, which are markers of EndMT. PCB153 also increased phosphorylation of ID3 in lung endothelial cells. To determine the molecular mechanism by which ID3 contributes to hyper-proliferative endothelial cells, we investigated ID3 transcriptional reprogramming using ChIP-Seq and RNA-Seq technology. We show here for the first time that ID3 is part of a more general mechanism of transcriptional regulation. Our ChIP-Seq data show that ID3 binds to a subset of approximately 1200 target genes. Comprehensive motif analysis of ChIP-Seq data using the MEME Suite software toolkit revealed that ID3 bound to the GAGAGAGAGA motif sequence on genomic DNA. We also show a significant preference of ID3 binding to motifs associated with transcription factors IRF1, BC11A, IRF4, PRDM1, FOXJ3, SMAD4, ZBTB6, GATA1, and STAT2. Using an integrative approach of ChIP-Seq and RNA-Seq data, we identified 19 genes whose promoter region was bound by ID3 and RNA was differentially expressed in ID3 overexpressing cells. In summary, our data demonstrated that PCB153 and/or ID3 induces proliferation of lung endothelial cells via transcriptional reprogramming. Discoveries from these findings will lay the necessary groundbreaking work for testing the efficacy of ID3 antagonists for the prevention and treatment of pathological vascular remodeling as well as provide a new paradigm by which PCBs may contribute to lung vascular toxicity.

This work analyzes the ecological, physical, emotional and health impacts of the US nuclear testing done in the Marshall Islands in the mid-20th century.