Jacqueline Chau

AG10 bacteriophages were preserved in pullulan-trehalose mixture as dried films and as coatings on food packaging. The phages encapsulated in pullulan-trehalose films were able to retain infectivity for up to 3 months at ambient storage conditions. Various buffers, disaccharides and disaccharide concentrations were investigated to optimize the long-term stability of the phages in the films. It was found that pullulan and trehalose need to be simultaneously present in the film to provide the stabilizing effect and that the presence of buffers that lead to the formation of crystals in the films must be avoided for phage activity to be maintained. Overall, this study describes a method of preserving bacteriophage activity in a dried format that has great potential for use as coatings, which can be used to create antimicrobial surfaces for food preparation and for food preservation.

Abstract The recent discovery of microRNAs (miRs) has added a new dimension to the mechanisms that regulate gene expression in normal cell development. Initial evidence also suggests that structural or functional alterations of miRs are associated with tumorigenesis. Nonetheless, the full set of genomic miRs is not known for any eukaryotic species including humans, since presently known miRs have been identified by virtue of their expression in only a few cell types. Analogous to other transcriptional units, it is conceivable that a significant part of the entire miR genomic pool may display tissue-specific expression. In order to understand the role of miRs in mature B cell function and lymphomagenesis, we planned the isolation of the full set of miRs (miRome) expressed in germinal center (GC) B cells and in GC-derived B cell lymphomas. Toward this end, we constructed libraries representative of all miRs expressed in normal GC B cells isolated from human tonsils and in a Burkitt lymphoma cell line (Ramos). Short RNAs were gel purified and linked to adaptor oligonucleotides, reverse transcribed, PCR amplified, cloned into an expression vector, and subjected to sequencing. Candidate miRs have been aligned to the human genome to retrieve their potential precursor sequences, whose folding characteristics have been analyzed in order to identify bona fide precursors. The cloned and structurally validated miR precursors have been matched to the public miR database to detect previously identified miR sequences. Initial results show that 453 miRs cloned from GC B cells correspond to 99 precursors (59 previously known and 40 newly discovered), while 233 miRs cloned from Ramos cell line identified 42 known and 46 new precursors. Overall, this initial analysis led to the discovery of 76 not previously reported miR precursors. Since less than a third of the available clones have been analyzed, it is expected that a significantly larger number of novel miRs will be identified by our method. A comprehensive analysis of miR expression profiles of normal versus neoplastic GC B cells will be obtained using a dedicated miR chip. Interestingly, initial results indicate that a set of miRs is expressed in normal GC cells, but not in Ramos and other GC-derived Burkitt lymphoma cell lines, suggesting that structural and/or functional alterations of miRs occur during lymphomagenesis.

Abstract MicroRNAs (miRNAs), an abundant class of ~22-nt endogenous regulatory RNAs that control gene expression at the posttranscriptional levels, have been implicated to play roles in the normal hematopoiesis and pathogenesis of leukemias. To identify “leukemic miRNAs”--miRNAs that may be oncogenes or tumor suppressors in human leukemias, we systematically cloned miRNAs from the blast cells of childhood acute lymphoblastic leukemia (ALL) patients with either a poor prognostic mixed-lineage leukemia rearrangement phenotype (MLL) or a prognostically more favorable precursor B-cell ALL phenotype (B-ALL). We have identified 87 known and 43 new human mature miRNAs, which potentially encoded by 101 known and 94 new human miRNA genes, respectively. Many newly identified miRNAs are not conserved in mouse, suggesting that systematic miRNA cloning analysis can facilitate the discovery of many novel human leukemia specific miRNAs. Interestingly, quantification of miRNA expression by real-time PCR analyses revealed that miRNAs are generally expressed at higher levels in MLL and B-ALL leukemia cells when compared to that in normal CD34+ bone marrow cells. We selected 21 highly differentially expression miRNA candidates and determined their expression in a larger group of 14 MLL patients and 16 B-ALL patients. We found that 9 miRNAs, including 4 newly identified miRNAs, were significantly differentially expressed (p < 0.05) between MLL and B-ALL subtypes. These findings demonstrate that the expression of both known and newly identified miRNA genes differs in genetically and prognostically different subgroups of ALL and normal CD34+ progenitor cells, suggesting that systematic miRNA cloning analyses and subsequent expression profiling analysis may facilitate the identification of specific signatures for leukemia classification and tumor suppressors or oncogenes that contribute to the pathogenesis of MLL and B-ALL.