Helicobacter pylori is a gastric bacterial pathogen that infects more than 50% of the world's population. H. pylori colonizes and persists in the harsh, acidic environment of the human stomach yet it is not an acidophile. The ability of H. pylori to successfully colonize the stomach is dependent on several factors. This includes helical shape that is thought to enhance initial colonization, urease-dependent ammonia production which works to neutralize the pH, and chemotaxis driven motility that enables the bacteria to navigate down the acidic gradient towards the neutral gastric mucosa, its preferred niche. Studies show that flagella-driven motility plays a key role in the pathogenesis of H. pylori; motile strains display higher infection rates and colonize the stomach longer in germ-free piglets compared to non-motile strains. Although the antrum serves as the preferential colonization site due to its lacking acid producing parietal cells, H. pylori can migrate and colonize other sites. In this project, we used a soft-agar assay to characterize the spreading motility of a collection of 42 H. pylori clones isolated from a single host at two different timepoints and from different gastric regions. Our results show that there are quantitative and qualitative differences in the spreading motility of these clinical isolates. The average halo diameters measured are collectively higher in the curved rod isolates from the earlier timepoint compared to helical and rod-shaped isolates from the later timepoint. The clones also display variable chemotactic patterns in soft agar assay with most strains producing concentric rings and a few strains forming uniform halos characteristic of nonchemotactic mutants. By assessing the differences observed, our study explores the factors that impact motility, such as flagellation and cell shape, in order to provide insight into these traits that best equip H. pylori to persist in the stomach. 

HOX genes encode for a group of homeodomain-containing transcription factors essential for patterning the skeleton in the developing embryo.  They are expressed in spatially restricted domains, where they regulate the patterning of specific bones that form within these expression domains.  The expression of Hox genes is continued into adulthood, however their function in the adult skeleton is relatively unknown.  We speculate that HOX genes regulate post-embryonic growth of bony elements which reside within their spatially-restricted expression domains.  In support of this hypothesis, we present three key findings.  First, we identify a subset of vertebrae in zebrafish (V2-V5) whose size and shape are under the control of hox5 genes.  These vertebrae reside in spatial domains associated with hox5 expression in embryonic zebrafish.  Second, we show that severe loss of hox5 induces gross changes in bone morphology, including loss of branching in the fourth ribs and sporadic fusion of the tripus with the fourth ribs.  Third, we note that fish with moderate loss of hox5 exhibit reduced size of bony elements but no obvious changes in bone morphology.  Our studies provide early evidence of the role of HOX genes in mediating post-embryonic growth of the skeleton.  They also help to elucidate potential vertebral homologies in zebrafish- an emerging model for orthapaedic research- and cervicothoracic vertebrae in mammals.

This research focuses on dissecting the molecular mechanism of cardiac regeneration in the animal model, zebrafish, upon a myocardial infarction like injury. Zebrafish are one of the few vertebrates that can fully regenerate their hearts after an injury in 30 days. This phenomenon is not seen in humans, who generate scar tissue after this injury with reduced circulatory efficiency. However, there is evidence that neonatal mice under 7 days old can regenerate their hearts, but this is lost upon adulthood. Determining this pathway is the first step to develop therapeutics in order to provide relief to people suffering from cardiac injuries. In this research, we used chemically ablated transgenic zebrafish to generate a 30% injury. We determined that upon an injury, both the Wnt pathway and the mTOR pathway are sequentially activated and upregulated to restart cardiac proliferation to regenerate the heart. Wnt pathway proteins like Axin and β-catenin are activated 3 days post injury and mTOR proteins like pS6 are activated gradually over 7 days post injury. The inhibition of the Wnt pathway using DKK showed a downregulation of the mTOR pathway and downregulation of cardiomyocyte proliferation. Inhibition of the mTOR pathway using Rapamycin also stopped cardiomyocyte proliferation from occurring. Mass spectrometry data showed a decrease in glutamine and an increase in leucine during the proliferative phase. Since leucine is one of the activators of the mTOR pathway, we see that the glutamine-leucine transporter is also upregulated post-injury. Thus, we show that heart regeneration in adult zebrafish occurs via cardiomyocyte proliferation by using the Wnt and mTOR pathways to upregulate cardiomyocyte proliferation upon injury.

The Polycomb Repressive Complex 2 (PRC2) is an important epigenetic remodeler in developmental transitions and cell fate determinations. PRC2 is responsible for the addition of H3K27me3 marks that repress developmental gene expression. The catalytic subunit of PRC2 is the methyltransferase (Enhancer of Zeste 2) EZH2 which binds to EED (Embryonic Ectoderm Development) to methylate H3K27 on gene promoter regions. To investigate the requirement of PRC2 in different developmental transitions, a computationally designed protein was utilized to inhibit EED-EZH2 interaction. The novel designed protein is named EED binder (EB) and competes over endogenous EZH2 on the EED binding cleft with 300 times greater affinity than endogenous EZH2. We cloned EB-GFP under heatshock inducible promoter and injected this construct to one cell zebrafish embryos to generate a germ line transmissible insertion. To study the requirement of PRC2 in early developing embryos (0-3dpf), we applied heatshock (HS) on EB-GFP positive and negative embryos. Western blot analysis revealed global downregulation of EZH2 and H3K27me3 in EB-GFP positive, but not control embryos. Additionally, Co-Immunoprecipitation experiments showed EB-GFP binding to EED. Finally, to test the requirement of PRC2 in caudal fin regeneration, adult (5 month old) EB-GFP positive and negative animals were fin-amputated and the regeneration growth rate was measured for 14 days. Our results show that EB-GFP positive fish were able to regenerate their fins faster, resulting in a large fin size compared to either negative or non-HS clutch mate. Overall, we have developed a computer designed inducible PRC2 inhibitory system to study PRC2 function in Zebrafish, at the whole animal level. In the future, we will utilize EB-GFP to explore PRC2 and other epigenetic modifiers that are required for tissue and organ regeneration before and after injury.

Humans are incapable of regenerating a majority of their major organs and tissues following traumatic injury, often resulting in an irreversible loss of function. Tadpoles of the frog genus Xenopus can regenerate multiple tissue types in response to injury, however this capability is lost after metamorphosis. This stage-specific regenerative capacity makes Xenopus a uniquely powerful model for studying factors that promote regeneration. Tadpoles develop ex-utero and do not develop mouths until days after fertilization. Before tadpoles are able to ingest exogenous food, they rely instead on maternal yolk stores for sustenance. A regenerative refractory period has been described in tadpoles of Xenopus laevis, in which regenerative capacity is transiently lost. In this study we describe a similar refractory period in the closely related Xenopus tropicalis, and observe that the onset of the refractory period aligns with the transition independent feeding. Based on this observation, we hypothesized that the lapse in regenerative capacity could be due to a lack of metabolic fuel. We used immunohistochemistry (IHC) against the yolk protein vitellogenin (vit) to study the utilization of maternal yolk stores during tadpole development. We find that yolk localization is dynamic over the course of development, and that it is ultimately is depleted by the onset of the refractory period. We additionally used IHC against phospho-Histone 3 (pH3), a marker of mitosis, to study proliferation during development and regeneration. We found that proliferation declines across development heading into the refractory period, in both uninjured and amputated contexts. Lastly, we successfully rescued both regeneration and proliferative rates by feeding tadpoles after they develop the ability to eat. As a whole, this work articulates that nutritive stress may contribute to the loss of regenerative capability in the refractory period, and that alleviation of this stress promotes regenerative ability in this context.

Epigenetic proteins modify the chromatin structure to manipulate gene expression, and dysregulated epigenetic modification in cells has been linked to cancer formation. Previous studies on young female Drosophila have shown that after injury, germline stem cells (GSC) are capable of entering and exiting a protective state called quiescence. When exposed to ionizing radiation (IR), the apoptotic differentiating daughter cells send a protective signal to GSC, resulting in GSC quiescence. This survival behavior of GSCs validates them as a potential model for cancer stem cells, which are a subset of tumor cells that are capable of withstanding traditional chemotherapy through reversible quiescence, resulting in future tumor relapse. To identify genes required for GSC survival, we performed a spatially restricted RNA interference (RNAi) screen. Here we show that two members of the repressive epigenetic regulator complex PRC1, Pc and Sce, are required for entry, while demethylase Utx is required for exit of GSC quiescence. Notably, PRC2 dependent H3K27me3 marks are required for PRC1 function, and Utx is required to erase these PRC2 dependent H3K27me3 marks. Importantly, we detected around a 3-fold increase in H3K27me3 marks in GSC following IR, suggesting that the repressive PRC1-PRC2 dependent complex is critical for entry, and elimination of PRC2 dependent marks is critical for exit from the quiescence state. Furthermore, we show that Trx, a writer enzyme which promotes euchromatin formation through H3K4me1 addition, is required for GSC exit from the quiescent. These data suggest that reversible quiescence in GSC is controlled by specific epigenetic states. In the future, more work is needed to investigate gene specificity of the epigenetic regulation.

Induced pluripotent stem cells (iPSC) have a high potential, for they can be differentiated into any cell type for regenerative medicine, drug discovery, developmental biology, and disease modeling. However, iPSC’s and their differentiated progeny display an undesired variability in their shape, contractile properties, growth rates, etc. Identifying subsets of phenotypes in iPSCs and their differentiated progeny will allow us to optimize tissue models for research. Here, we generated a rainbow reporter line in iPSCs that can track individual cells as they clonally expand and differentiate while providing phenotypic information. Knocking in four copies of a cassette containing three distinct fluorescent proteins allowed the expression of up to eighteen different colors. However, not all colors were present in equal proportion, increasing the probability that distinct lineages could have the same color. To achieve an equal color distribution, colored cells were isolated by sparsely plating a culture of mixed colored cells. After a week of expansion, individual colonies were picked and imaged under a spinning disk microscope to determine the color of the colony and whether it was single lineage or mixed. Viable cell lines were isolated and frozen in stock. These cells will be examined for markers of cell proliferation, pluripotency, apoptosis and quantitative RNA expression analysis to confirm that the color barcoded iPSCs act the same as non-engineered iPSCs. To date, we were able to create eight color barcoded iPSC lines for further experimentation, increasing the concentration limit of colored cells in non-colored cells by five-fold. The next step will engineer 3D tissues by growing iPSC-derived cardiac cells in a mold to simulate in vivo tissue development. Colored coded cells will allow us to track how the initial location/physical stresses/phenotype of an iPSC-derived cardiac cell in an engineered tissue determines its tissue layer and cell type.

Over the past fifty years or so, scientists have successfully isolated microorganisms, known as endophytes, from inside nearly every region of the plant, including the roots, stems, and leaves. Some of these microorganisms have the ability to fix atmospheric nitrogen, and have even been shown to confer an array of additional benefits to their plant hosts, ranging from fungal pathogen resistance to increased stress tolerance, and more. Recently, many scientists conducting endophyte research have observed their endophytes losing their plant-enhancing activity after extended periods of isolation from their original plant hosts. This makes the plant-enhancing effects of the isolated endophytes impossible to study, and more importantly points to a crucial need for a better understanding of plant-endophyte communication mechanisms. Because reactivation of several endophytes was observed after introducing native plant extract back into the isolated endophytes’ medium, we have embarked on a journey to determine what in the plant medium causes these inactive endophytes to regain their plant-enhancing abilities. To do this, we have first determined the level (transcriptional, translational, or post-translational) at which nitrogen-fixation, a crucial symbiotic activity, is regulated by the presence of plant extract in one of our endophytes. We then designed and built a reporter plasmid to track the expression of key nitrogen-fixing genes, and will next fractionate the native plant extract and use this reporter plasmid to monitor the effects of different plant fractions. This will hopefully lead to key insights into the potential plant signal that initiates many symbiotic endophyte activities. The exploration of the plant-endophyte symbiotic signaling system has the potential to impact the fields of agriculture, forestry, and environmental sciences worldwide.