Preparation of a bank of cloned genes from the chromosome of Agrobacterium tumefaciens and the isolation of genes involved in DNA repair and genetic recombination

The virulent property of the Agrobacterium tumefaciens is associated with its tumor inducing (Ti) plasmid. Recent studies on the virulence of this bacterium has shown that genes located on its chromosome also contribute to this property. One such gene is thought to produce a protein that has properties similar to that of the recA protein of E. colt. This thesis outlines the techniques that were used to try and isolate the recA-like gene from the chromosome of the Agrobacterium tumefaciens. All the techniques used in this study are outlined in detail and an explanation given for the choice of each technique. For reasons not completely understood, we were unable to isolate the recA-like gene even though a gene bank was successfully constructed and appropriately sized fragments extracted. Attempts were made to explain some of the unexpected results and appropriate steps were proposed to further understand them

H19 Noncoding RNA, an Independent Prognostic Factor, Regulates Essential Rb-E2F and CDK8-β-Catenin Signaling in Colorectal Cancer

The clinical significance of long noncoding RNAs (lncRNAs) in colorectal cancer (CRC) remains largely unexplored. Here, we analyzed a large panel of lncRNA candidates with The Cancer Genome Atlas (TCGA) CRC dataset, and identified H19 as the most significant lncRNA associated with CRC patient survival. We further validated such association in two independent CRC cohorts. H19 silencing blocked G1-S transition, reduced cell proliferation, and inhibited cell migration. We profiled gene expression changes to gain mechanism insight of H19 function. Transcriptome data analysis revealed not only previously identified mechanisms such as Let-7 regulation by H19, but also RB1-E2F1 function and β-catenin activity as essential upstream regulators mediating H19 function. Our experimental data showed that H19 affects phosphorylation of RB1 protein by regulating gene expression of CDK4 and CCND1. We further demonstrated that reduced CDK8 expression underlies changes of β-catenin activity, and identified that H19 interacts with macroH2A, an essential regulator of CDK8 gene transcription. However, the relevance of H19-macroH2A interaction in CDK8 regulation remains to be experimentally determined. We further explored the clinical relevance of above mechanisms in clinical samples, and showed that combined analysis of H19 with its targets improved prognostic value of H19 in CRC

MiR-543 regulates the epigenetic landscape of myelofibrosis by targeting TET1 and TET2

Myelofibros is (MF) is a myeloproliferative neoplasm characterized by cytopenia and extramedullary hematopoiesis, resulting in splenomegaly. Multiple pathological mechanisms (e.g., circulating cytokines and genetic alterations, such as JAK(V617F) mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK(V617F) inhibitor therapy remains unknown. Among MF patients who were treated with the JAK inhibitor ruxolitinib, we compared noncoding RNA profiles of ruxolitinib therapy responders versus nonresponders and found miR-S43 was significantly upregulated in non responders. We validated these findings by reverse transcription-quantitative PCR. in this same cohort, in 2 additional independent MF patient cohorts from the United States and Romania, and in a JAK2(V617F) mouse model of MF. Both in vitro and in vivo models were used to determine the underlying molecular mechanism of miR-543 in MF. Here, we demonstrate that miR-543 targets the dioxygenases ten-eleven translocation 1 (TET1) and 2 (TET2) in patients and in vitro, causing increased levels of global 5-methylcytosine, while decreasing the acetylation of histone 3, STAT3, and tumor protein p53. Mechanistically, we found that activation of STAT3 by JAKs epigenetically controls miR-543 expression via binding the promoter region of miR-543. Furthermore, miR-543 upregulation promotes the expression of genes related to drug metabolism, including CYP3A4, which is involved in ruxolitinib metabolism. Our findings suggest miR-543 as a potentially novel biomarker for the prognosis of MF patients with a high risk of treatment resistance and as a potentially new target for the development of new treatment options

Activation of a novel Bcr/Abl destruction pathway by WP1130 induces apoptosis of chronic myelogenous leukemia cells

Imatinib mesylate (Gleevec) is effective therapy against Philadelphia chromosome–positive leukemia, but resistance develops in all phases of the disease. Bcr/Abl point mutations and other alterations reduce the kinase inhibitory activity of imatinib mesylate; thus, agents that target Bcr/Abl through unique mechanisms may be needed. Here we describe the activity of WP1130, a small molecule that specifically and rapidly down-regulates both wild-type and mutant Bcr/Abl protein without affecting bcr/abl gene expression in chronic myelogenous leukemia (CML) cells. Loss of Bcr/Abl protein correlated with the onset of apoptosis and reduced phosphorylation of Bcr/Abl substrates. WP1130 did not affect Hsp90/Hsp70 ratios within the cells and did not require the participation of the proteasomal pathway for loss of Bcr/Abl protein. WP1130 was more effective in reducing leukemic versus normal hematopoietic colony formation and strongly inhibited colony formation of cells derived from patients with T315I mutant Bcr/Abl–expressing CML in blast crisis. WP1130 suppressed the growth of K562 heterotransplanted tumors as well as both wild-type Bcr/Abl and T315I mutant Bcr/Abl–expressing BaF/3 cells transplanted into nude mice. Collectively, our results demonstrate that WP1130 reduces wild-type and T315I mutant Bcr/Abl protein levels in CML cells through a unique mechanism and may be useful in treating CML

Benzophenone and Fimetarone Derivatives from the Coprophilous Fungus <i>Delitschia confertaspora</i>

Studies of the genome-sequenced, flutimide-producing coprophilous fungus <i>Delitschia confertaspora</i> (ATCC 74209), originally obtained from a sample of rock hyrax (<i>Procavia capensis</i>) dung collected in Namibia, led to the discovery of three new highly aromatic natural products named delicoferones A–B (<b>1</b>–<b>2</b>) and fimetarone B (<b>3</b>). The new benzophenone derivatives <b>1</b> and <b>2</b> have a somewhat unusual skeleton that incorporates three aromatic rings linked via two ketone carbonyl groups, while <b>3</b> contains a spiro­[chroman-3,7′-isochromene]-4,6′(8′H) skeleton reported only once previously. The structures of these compounds were assigned mainly by analysis of 2D NMR and HRESITOFMS data

3D tissue-engineered model of Ewing's sarcoma

Despite longstanding reliance upon monolayer culture for studying cancer cells, and numerous advantages from both a practical and experimental standpoint, a growing body of evidence suggests that more complex three-dimensional (3D) models are necessary to properly mimic many of the critical hallmarks associated with the oncogenesis, maintenance and spread of Ewing's sarcoma (ES), the second most common pediatric bone tumor. And as clinicians increasingly turn to biologically-targeted therapies that exert their effects not only on the tumor cells themselves, but also on the surrounding extracellular matrix, it is especially important that preclinical models evolve in parallel to reliably measure antineoplastic effects and possible mechanisms of de novo and acquired drug resistance. Herein, we highlight a number of innovative methods used to fabricate biomimetic ES tumors, encompassing both the surrounding cellular milieu and the extracellular matrix (ECM), and suggest potential applications to advance our understanding of ES biology, preclinical drug testing, and personalized medicine

Anti-<i>Cryptococcus</i> Phenalenones and Cyclic Tetrapeptides from <i>Auxarthron pseudauxarthron</i>

Auxarthrones A–E (<b>1</b>–<b>5</b>), five new phenalenones, and two new naturally occurring cyclic tetrapeptides, auxarthrides A (<b>7</b>) and B (<b>8</b>), were obtained from three different solvent extracts of cultures of the coprophilous fungus <i>Auxarthron pseudauxarthron</i>. Auxarthrones C (<b>3</b>) and E (<b>5</b>) possess an unusual 7a,8-dihydrocyclopenta­[<i>a</i>]­phenalene-7,9-dione ring system that has not been previously observed in natural products. Formation of <b>1</b>–<b>5</b> was found to be dependent on the solvent used for culture extraction. The structures of these new compounds were elucidated primarily by analysis of NMR and MS data. Auxarthrone A (<b>1</b>) was obtained as a mixture of chromatographically inseparable racemic diastereomers (<b>1a</b> and <b>1b</b>) that cocrystallized, enabling confirmation of their structures by X-ray crystallography. The absolute configurations of <b>7</b> and <b>8</b> were assigned by analysis of their acid hydrolysates using Marfey’s method. Compound <b>1</b> displayed moderate antifungal activity against <i>Cryptococcus neoformans</i> and <i>Candida albicans</i>, but did not affect human cancer cell lines

Anti-<i>Cryptococcus</i> Phenalenones and Cyclic Tetrapeptides from <i>Auxarthron pseudauxarthron</i>

Auxarthrones A–E (<b>1</b>–<b>5</b>), five new phenalenones, and two new naturally occurring cyclic tetrapeptides, auxarthrides A (<b>7</b>) and B (<b>8</b>), were obtained from three different solvent extracts of cultures of the coprophilous fungus <i>Auxarthron pseudauxarthron</i>. Auxarthrones C (<b>3</b>) and E (<b>5</b>) possess an unusual 7a,8-dihydrocyclopenta­[<i>a</i>]­phenalene-7,9-dione ring system that has not been previously observed in natural products. Formation of <b>1</b>–<b>5</b> was found to be dependent on the solvent used for culture extraction. The structures of these new compounds were elucidated primarily by analysis of NMR and MS data. Auxarthrone A (<b>1</b>) was obtained as a mixture of chromatographically inseparable racemic diastereomers (<b>1a</b> and <b>1b</b>) that cocrystallized, enabling confirmation of their structures by X-ray crystallography. The absolute configurations of <b>7</b> and <b>8</b> were assigned by analysis of their acid hydrolysates using Marfey’s method. Compound <b>1</b> displayed moderate antifungal activity against <i>Cryptococcus neoformans</i> and <i>Candida albicans</i>, but did not affect human cancer cell lines