There are not enough donor lungs available to meet the increasing demand for lung transplantation. To compound the problem, transplant recipients have a projected survival time of only 5.7 years despite life-long immunosuppression. An alternative approach for acquiring transplantable lungs and reducing post-operative complications may be possible through tissue engineering. Perfusion-decellularization generates natural, three-dimensional extracellular matrix (ECM) scaffolds of an organ that are apt for tissue engineering. Decellularization of the heart, lung, liver, kidney, and pancreas has been reported in animal models and from human tissue. Decellularization of fibrotic and emphysematic lungs indicated that this technique can efficiently remove cells from diseased tissue—a potential source of materials for engineering of transplantable lung tissue. Pulmonary hypertension (PHT) is a vascular disease characterized by increased pulmonary vascular resistance leading to right heart failure and death. Lungs damaged by PHT are unsuitable for transplantation; however, decellularization of these organs may provide scaffolds appropriate for ex vivo lung engineering. Monocrotaline-induced PHT (MCT-PHT) is a well-established model of this disease in rats closely resembling the clinical presentation of PHT in humans. Thus, decellularization and recellularization of hypertensive lungs was evaluated using the MCT-PHT model. Decellularization of control and MCT-PHT Sprague-Dawley rat lungs was accomplished by treating the lungs with Triton X-100, sodium deoxycholate (SDC), NaCl, and DNase. The resulting acellular matrices were extensively characterized by molecular, mechanical, and structural analyses revealing that decellularization was able to remove cells while leaving the ECM components and lung ultrastructure intact; however, the vasculature of MCT-PHT acellular lung scaffolds was narrower than control scaffolds—a hallmark of PHT. To evaluate the effect of narrowed vasculature on the use of hypertensive lungs for tissue engineering, an optimal vascular recellularization technique was developed. Gravity-based seeding of endothelial cells followed by bioreactor-based whole-organ culture resulted in efficient vascular recellularization of control lung scaffolds. However, this method led to heterogeneous re-endothelialization of the vasculature of MCT-PHT matrices suggesting that additional manipulation or optimization is required. / acase@tulane.edu

The active human retrotransposon L1 is the most prevalent human retroelement, constituting 17% of the mass of the human genome and contributing significantly to mutagenesis. L1 mutagenizes human genomes in a number of ways including insertional mutagenesis of itself and other retrotransposons, creating of DNA double strand breaks, and induction of non-allelic homologous recombination. Through these processes, the activity of L1 is responsible for approximately 0.5% of all new genetic diseases. All L1-derived mutagenesis stems from the activity of a small number of intact full-length L1 loci that remain capable of mobilization. A smaller subset of these active L1s are called hot L1s and are responsible for the vast majority of all L1 activity. Hot L1s are polymorphic in the population and represent evolutionarily recent L1 insertion events. Here, we show that potentially active full length L1 elements are more prevalent in individual genomes than previously believed. We find that the typical individual likely harbors approximately 60 active and 50 hot L1s. However, we also find that there is significant variation between individuals in numbers of potentially active L1s. As a result, the mutagenic burden associated with L1 likely varies between individuals. / acase@tulane.edu

acase@tulane.edu

MicroRNAs (miRNAs) are small non-coding 18-22 long RNAs, which act as key mediators in many cellular processes involved in tumorigenesis including proliferation, differentiation, invasion, and apoptosis. miRNAs repress gene expression by inhibiting mRNA translation or by promoting mRNA degradation. Due to the importance of miRNAs as master regulators of gene expression many studies have emerged to distinguish both miRNA targets and regulatory mechanisms governing miRNA translation and maturation. Uncovering mechanisms that govern miRNA expression and function in cellular biology gives greater insight into pathways facilitating multiple cellular processes involved in tumor initiation and progression. miRNAs are altered in breast cancer and these alterations can lead to changes in signaling pathways involved in cancer progression. The purpose of this work is to determine the effects of miRNA expression on the cancer phenotype and to examine the roles of insulin-like growth factor 1 (IGF-1) and estrogen receptor (ER) signaling on miRNA expression and function in ER+ breast cancer cell systems. Our results demonstrate the intricate relationship between miRNAs and cell signaling networks. Through uncovering miRNA regulating networks such as IGF-1 signaling and ER induced miRNA maturation and star strand selection we have added greater insight to the complex mechanisms involved in miRNA function. miRNAs function through inhibition of mRNA targets, though the possible targets for a single miRNA can be in the thousands. Additionally, mRNA isoform variability and loss of 3’UTR greatly enhance the complexity of these networks. / acase@tulane.edu

MicroRNAs (miRNAs) are a group of small, noncoding RNAs that modulate the translation of genes into proteins by binding to specific target sites in messenger RNAs. This study investigated the biological function and molecular mechanism of microRNA-21 (miR-21) in human cholangiocarcinoma. In situ hybridization analysis of human cholangiocarcinoma tissues showed increased miR-21 in cholangiocarcinoma cells compared to the noncancerous biliary epithelial cells. Forced overexpression of miR-21 by lentivirus transduction enhanced human cholangiocarcinoma cell growth and clonogenic efficiency in vitro, whereas inhibition of miR-21 decreased these parameters. MiR-21 overexpression also promoted cholangiocarcinoma growth in a tumor xenograft model. The NAD+-linked 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme that converts the pro-tumorigenic prostaglandin E2 (PGE2) to biologically inactive metabolite, was identified as a direct target of miR-21 in cholangiocarcinoma cells. In parallel, cyclooxygenase-2 (COX-2) overexpression and PGE2 treatment increased miR-21 expression and enhanced miR-21 promoter reporter activity in human cholangiocarcinoma cells. Our results disclose a novel cross-talk between COX-2/PGE2 and miR-21 signaling pathways that converges at 15-PGDH which is crucial in cholangiocarcinogenesis and tumor progression. The enhancer of zeste homolog 2 (EZH2) is the catalytic subunit in the PRC2 complex catalyzing the trimethylation of histone3 lysine27 (H3K27) and mediates gene silencing of the target genes. The biological function of EZH2 in cholangiocarcinoma was investigated in this study. Immunohistochemistry staining of EZH2 on human cholangiocarcinoma tissues showed increased EZH2 expression in cholangiocarcinoma cells. Pharmacologically inhibition of EZH2 by EZH2 inhibitors decreased cholangiocarcinoma growth and induced G1 arrest. The CD133, one of the putative cancer stem cell markers, was found to express in the cholangiocarcinoma cell lines we used. Inhibition of EZH2 decreased CD133+ population and the sphere forming ability of cancer cells. Our results indicate that EZH2 may represent a promising target for targeting the tumor-initiating cell population and future cholangiocarcinoma therapy. / acase@tulane.edu

acase@tulane.edu

acase@tulane.edu

No description available.

In the work presented here we have used a function-based approach to isolate 12 novel cell-penetrating peptides from a 10,000+ member peptide library of rational design. Our unique high-throughput screen differentiates non-membranolytic from membranolytic translocation of peptides across lipid bilayers, thus allowing the selection of potential cell-penetrating peptides over potential antimicrobial peptides or peptide toxins. The 12 residue framework of the peptide library, designed with translocation in mind, is a series of 9 combinatorial sites followed by a C-terminal α-1-chymotrypsin cleavage site that is integral to the screen. The resulting residue in each of the combinatorial sites is one of 2 - 4 variable amino acids, with a hydrophobic or cationic residue available in each position. The sequences of nonpore-forming translocating peptides pulled from the screen have a 3 residue motif, Leu-Leu-Arg (p=10-5), and an overall under representation of basic residues in favor of hydrophobic amino acids. Upon characterization, these novel peptides were shown to behave akin to known cell-penetrating peptides found in nature. Ex vivo studies, in mammalian tissue cultures, revealed that the peptides translocate across the cell membrane without toxicity to the cell. In addition, structural studies showed a lack of convergence regarding a structure- function relationship, a continued trend seen among membrane-active peptides. In the course of the screen and the ex vivo studies, the peptides carried small polar molecules across lipid bilayers and biological membranes respectively; suggesting that, in addition to being cell-penetrating peptides, they could be put to use as effective therapeutic agents. The discovery of these novel cell-penetrating peptides by use of our screen supports function-based screening of peptide libraries as the best way to arrive at de novo membrane-active peptides with specific functions of interest. / acase@tulane.edu

Background: Chronic wounds are a serious medical condition affecting over 6 million people in the United States. Biofilms, which are communities of bacteria attached to a surface and protected by a polysaccharide coating, are intimately associated with the development of chronic wounds. They alter the host immune response and establish a microenvironment that prevents wound healing. Current treatment options do not target biofilms. One novel treatment solution for chronic wounds involves the paracrine factors from mesenchymal stem cells (MSCs), which have been shown to stimulate wound healing in non-infected wounds. The aim of this dissertation was to examine the effects of reprogrammed MSCs and their paracrine factors on ameliorating infection and accelerating wound closure in biofilm infected wounds. Methods: MSCs were reprogrammed by 3 strategies: seeding on an extracellular matrix (ECM), as spheroids in static culture, and as spheroids in a bioreactor. The paracrine factors were analyzed using a 14-plex cytokine assay to confirm changes from baseline. The paracrine factors were applied to mature P. aeruginosa biofilms in vitro and the number of viable bacteria were quantitated. They were also used to stimulate RAW 264.7 murine macrophages and analyzed for the presence of CD206, a marker of anti-inflammatory macrophage phenotype. BALB/cJ mice were wounded and infected with P. aeruginosa biofilms and the paracrine factors from reprogrammed MSCs were applied topically. The wound area and CFU counts of the treatment group were compared to the control and untreated groups. Results: MSCs grown as spheroids in a bioreactor produced significant increases in IL-6 and IL-8 after 3 and 7 days (p<0.05 and p<0.0001). The paracrine factors from MSCs grown on ECM were found to reduce P. aeruginosa biofilm growth significantly (p<0.01). Spheroids grown statically and in a bioreactor increased the amount of macrophages expressing CD206. Mice wounds receiving the paracrine factors from MSCs grown on ECM had lower bacterial counts and an increased rate of wound closure compared to non-treated mice wounds. Conclusions: The results indicate that paracrine factors from reprogrammed MSCs accelerated wound healing and reduced the bacterial burden in biofilm infected wounds. / acase@tulane.edu