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

Study of the role of the Myxococcus xanthus ESG locus in development and lipid biosynthesis.

Analysis of the pattern of expression of developmentally regulated genes in the esg mutant background, suggests that E-signaling dependent events of development occur after 3 to 5 hours of development. It has been predicted that iso-15:0 may function as a signal molecule based on the observation that esg mutants do not develop and synthesize low levels of iso-15:0. By growing wild-type and esg mutant cells of M. xanthus with H\sp3-labeled branched-chain fatty acids we showed that esg mutant cells incorporated labeled leucine into lipids at no more than 11% of the wild-type cells. Levels of iso-15:0 incorporated into the phospholipids of the esg mutant cells were observed to be more than three times lower than those of wild-type cells. These results confirmed that the reduced synthesis of iso-15:0 in the esg mutant may be responsible for its developmental defect. If iso-15:0 is a signal molecule we predict that its levels would increase during the early stages of development. Analysis of leucine labeled free fatty acid fractions purified from developing wild-type cells showed that iso-15:0 is not released as a free fatty acid but is a component of a larger labeled species whose level remained relatively constant during the first ten hours of development. These labeled lipid species were biologically active and rescued development of an esg mutant. Iso-15:0 by itself was also biologically active. We propose that fatty acids or a combination of fatty acids and other lipid compounds synthesized during vegetative growth in the esg-dependent pathway may function as signal molecules during development of M. xanthus. A nutritional dependent fatty acid response occurs in M. xanthus. We observed that esg gene expression and BCKAD activity were under nutritional regulation and was responsible for the changing fatty acid composition of the cell. Esg mutants failed to respond to nutritional changes. We conclude that the BCKAD is required for the nutrition dependent fatty acid response in M. xanthus

Carbon Nanotubes: Solution for the Therapeutic Delivery of siRNA?

Carbon nanotubes have many unique physical and chemical properties that are being widely explored for potential applications in biomedicine especially as transporters of drugs, proteins, DNA and RNA into cells. Specifically, single-walled carbon nanotubes (SWCNT) have been shown to deliver siRNA to tumors in vivo. The low toxicity, the excellent membrane penetration ability, the protection afforded against blood breakdown of the siRNA payload and the good biological activity seen in vivo suggests that SWCNT may become universal transfection vehicles for siRNA and other RNAs for therapeutic applications. This paper will introduce a short review of a number of therapeutic applications for carbon nanotubes and provide recent data suggesting SWCNT are an excellent option for the delivery of siRNA clinically

Expression of histone deacetylase (HDAC) family members in bortezomib-refractory multiple myeloma and modulation by panobinostat

Aim: Multiple myeloma (MM) is a hematological malignancy of antibody-producing mature B cells or plasma cells. The proteasome inhibitor, bortezomib, was the first-in-class compound to be FDA approved for MM and is frequently utilized in induction therapy. However, bortezomib refractory disease is a major clinical concern, and the efficacy of the pan-histone deacetylase inhibitor (HDACi), panobinostat, in bortezomib refractory disease indicates that HDAC targeting is a viable strategy. Here, we utilized isogenic bortezomib resistant models to profile HDAC expression and define baseline and HDACi-induced expression patterns of individual HDAC family members in sensitive vs. resistant cells to better understanding the potential for targeting these enzymes.Methods: Gene expression of HDAC family members in two sets of isogenic bortezomib sensitive or resistant myeloma cell lines was examined. These cell lines were subsequently treated with HDAC inhibitors: panobinostat or vorinostat, and HDAC expression was evaluated. CRISPR/Cas9 knockdown and pharmacological inhibition of specific HDAC family members were conducted.Results: Interestingly, HDAC6 and HDAC7 were significantly upregulated and downregulated, respectively, in bortezomib-resistant cells. Panobinostat was effective at inducing cell death in these lines and modulated HDAC expression in cell lines and patient samples. Knockdown of HDAC7 inhibited cell growth while pharmacologically inhibiting HDAC6 augmented cell death by panobinostat.Conclusion: Our data revealed heterogeneous expression of individual HDACs in bortezomib sensitive vs. resistant isogenic cell lines and patient samples treated with panobinostat. Cumulatively our findings highlight distinct roles for HDAC6 and HDAC7 in regulating cell death in the context of bortezomib resistance