Integrated genome-wide chromatin occupancy and expression analyses identify key myeloid pro-differentiation transcription factors repressed by Myb

To gain insight into the mechanisms by which the Myb transcription factor controls normal hematopoiesis and particularly, how it contributes to leukemogenesis, we mapped the genome-wide occupancy of Myb by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) in ERMYB myeloid progenitor cells. By integrating the genome occupancy data with whole genome expression profiling data, we identified a Myb-regulated transcriptional program. Gene signatures for leukemia stem cells, normal hematopoietic stem/progenitor cells and myeloid development were overrepresented in 2368 Myb regulated genes. Of these, Myb bound directly near or within 793 genes. Myb directly activates some genes known critical in maintaining hematopoietic stem cells, such as Gfi1 and Cited2. Importantly, we also show that, despite being usually considered as a transactivator, Myb also functions to repress approximately half of its direct targets, including several key regulators of myeloid differentiation, such as Sfpi1 (also known as Pu.1), Runx1, Junb and Cebpb. Furthermore, our results demonstrate that interaction with p300, an established coactivator for Myb, is unexpectedly required for Myb-mediated transcriptional repression. We propose that the repression of the above mentioned key pro-differentiation factors may contribute essentially to Myb's ability to suppress differentiation and promote self-renewal, thus maintaining progenitor cells in an undifferentiated state and promoting leukemic transformation. © 2011 The Author(s).

Cloning and gastrointestinal expression of rat hephaestin: Relationship to other iron transport proteins

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.

Heterogeneity of miR-10b expression in circulating tumor cells

Circulating tumor cells (CTCs) in the blood of cancer patients are recognized as important potential targets for future anticancer therapies. As mediators of metastatic spread, CTCs are also promising to be used as € liquid biopsyto aid clinical decision-making. Recent work has revealed potentially important genotypic and phenotypic heterogeneity within CTC populations, even within the same patient. MicroRNAs (miRNAs) are key regulators of gene expression and have emerged as potentially important diagnostic markers and targets for anti-cancer therapy. Here, we describe a robust in situ hybridization (ISH) protocol, incorporating the CellSearch ® CTC detection system, enabling clinical investigation of important miRNAs, such as miR-10b on a cell by cell basis. We also use this method to demonstrate heterogeneity of such as miR-10b on a cell-by-cell basis. We also use this method to demonstrate heterogeneity of miR-10b in individual CTCs from breast, prostate and colorectal cancer patients.

Analysis of differential gene expression based on Bayesian estimation of variance

Gene expression is arguably the most important indicator of biological function. Thus identifying differentially expressed genes is one of the main aims of high throughout studies that use microarray and RNAseq platforms to study deregulated cellular pathways. There are many tools for analysing differentia gene expression from transciptomic datasets. The major challenge of this topic is to estimate gene expression variance due to the high amount of ‘background noise’ that is generated from biological equipment and the lack of biological replicates. Bayesian inference has been widely used in the bioinformatics field. In this work, we reveal that the prior knowledge employed in the Bayesian framework also helps to improve the accuracy of differential gene expression analysis when using a small number of replicates. We have developed a differential analysis tool that uses Bayesian estimation of the variance of gene expression for use with small numbers of biological replicates. Our method is more consistent when compared to the widely used cyber-t tool that successfully introduced the Bayesian framework to differential analysis. We also provide a user-friendly web based Graphic User Interface for biologists to use with microarray and RNAseq data. Bayesian inference can compensate for the instability of variance caused when using a small number of biological replicates by using pseudo replicates as prior knowledge. We also show that our new strategy to select pseudo replicates will improve the performance of the analysis. - See more at: http://www.eurekaselect.com/node/138761/article#sthash.VeK9xl5k.dpuf

Development of molecular tools for the improvement of transgene expression in sugar cane

Biotechnology has the potential to improve sugar cane, one of the world's major crops for food and fuel. This research describes the detailed characterisation of introns and their potential for enhancing transgene expression in sugar cane via intron-mediated enhancement (IME). IME is a phenomenon whereby an intron enhances gene expression from a promoter. Current knowledge on the mechanism of IME or its potential for enhancing gene expression in sugar cane is limited. A better understanding of the factors responsible for IME will help develop new molecular tools that facilitate high levels of constitutive and tissue-specific gene expression in this crop.

Urinary tract infection of mice to model human disease: Practicalities, implications and limitations

Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Murine models of human UTI are vital experimental tools that have helped to elucidate UTI pathogenesis and advance knowledge of potential treatment and infection prevention strategies. Fundamentally, several variables are inherent in different murine models, and understanding the limitations of these variables provides an opportunity to understand how models may be best applied to research aimed at mimicking human disease. In this review, we discuss variables inherent in murine UTI model studies and how these affect model usage, data analysis and data interpretation. We examine recent studies that have elucidated UTI host–pathogen interactions from the perspective of gene expression, and review new studies of biofilm and UTI preventative approaches. We also consider potential standards for variables inherent in murine UTI models and discuss how these might expand the utility of models for mimicking human disease and uncovering new aspects of pathogenesis

Genome-wide mapping of cystitis due to Streptococcus agalactiae and Escherichia coli in mice identifies a unique bladder transcriptome that signifies pathogen-specific antimicrobial defense against urinary tract infection

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35 year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; qRT-PCR was used to analyze selected gene responses identified in array datasets. A surprisingly small significant gene list of 172 genes was identified at 24h; this compared to 2507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2h. Bioinformatics analyses including integrative systems-level network mapping revealed multiple activated biological pathways in the GBS cystitis transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens.

Anti-bacterial surfaces: Natural agents, mechanisms of action, and plasma surface modification

Strategies that confine antibacterial and/or antifouling property to the surface of the implant, by modifying the surface chemistry and morphology or by encapsulating the material in an antibiotic-loaded coating, are most promising as they do not alter bulk integrity of the material. Among them, plasma-assisted modification and catechol chemistry stand out for their ability to modify a wide range of substrates. By controlling processing parameters, plasma environment can be used for surface nano structuring, chemical activation, and deposition of biologically active and passive coatings. Catechol chemistry can be used for material-independent, highly-controlled surface immobilisation of active molecules and fabrication of biodegradable drug-loaded hydrogel coatings. In this article, we comprehensively review the role plasma-assisted processing and catechol chemistry can play in combating bacterial colonisation on medically relevant coatings, and how these strategies can be coupled with the use of natural antimicrobial agents to produce synthetic antibiotic-free antibacterial surfaces.

Plasma assisted surface modification of organic biopolymers to prevent bacterial attachment

Despite many synthetic biomaterials having physical properties that are comparable or even superior to those of natural body tissues, they frequently fail due to the adverse physiological reactions they cause within the human body, such as infection and inflammation. The surface modification of biomaterials is an economical and effective method by which biocompatibility and biofunctionality can be achieved while preserving the favorable bulk characteristics of the biomaterial, such as strength and inertness. Amongst the numerous surface modification techniques available, plasma surface modification affords device manufacturers a flexible and environmentally friendly process that enables tailoring of the surface morphology, structure, composition, and properties of the material to a specific need. There are a vast range of possible applications of plasma modification in biomaterial applications, however, the focus of this review paper is on processes that can be used to develop surface morphologies and chemical structures for the prevention of adhesion and proliferation of pathogenic bacteria on the surfaces of in-dwelling medical devices. As such, the fundamental principles of bacterial cell attachment and biofilm formation are also discussed. Functional organic plasma polymerised coatings are also discussed for their potential as biosensitive interfaces, connecting inorganic/metallic electronic devices with their physiological environments.

The effect of polyterpenol thin film surfaces on bacterial viability and adhesion

The nanometer scale surface topography of a solid substrate is known to influence the extent of bacterial attachment and their subsequent proliferation to form biofilms. As an extension of our previous work on the development of a novel organic polymer coating for the prevention of growth of medically significant bacteria on three-dimensional solid surfaces, this study examines the effect of surface coating on the adhesion and proliferation tendencies of Staphylococcus aureus and compares to those previously investigated tendencies of Pseudomonas aeruginosa on similar coatings. Radio frequency plasma enhanced chemical vapor deposition was used to coat the surface of the substrate with thin film of terpinen-4-ol, a constituent of tea-tree oil known to inhibit the growth of a broad range of bacteria. The presence of the coating decreased the substrate surface roughness from approximately 2.1 nm to 0.4 nm. Similar to P. aeruginosa, S. aureus presented notably different patterns of attachment in response to the presence of the surface film, where the amount of attachment, extracellular polymeric substance production, and cell proliferation on the coated surface was found to be greatly reduced compared to that obtained on the unmodified surface. This work suggests that the antimicrobial and antifouling coating used in this study could be effectively integrated into medical and other clinically relevant devices to prevent bacterial growth and to minimize bacteria-associated adverse host responses.

Bacterial extracellular polysaccharides

Extracellular polysaccharides are as structurally and functionally diverse as the bacteria that synthesise them. They can be present in many forms, including cell-bound capsular polysaccharides, unbound "slime", and as O-antigen component of lipopolysaccharide, with an equally wide range of biological functions. These include resistance to desiccation, protection against nonspecific and specific host immunity, and adherence. Unsurprisingly then, much effort has been made to catalogue the enormous structural complexity of the extracellular polysaccharides made possible by the wide assortment of available monosaccharide combinations, non-carbohydrate residues, and linkage types, and to elucidate their biosynthesis and export. In addition, the work is driven by the commercial potential of these microbial substances in food, pharmaceutics and biomedical industries. Most recently, bacteria-mediated environmental restoration and bioleaching have been attracting much attention owing to their potential to remediate environmental effluents produced by the mining and metallurgy industries. In spite of technological advances in chemistry, molecular biology and imaging techniques that allowed for considerable expansion of knowledge pertaining to the bacterial surface polysaccharides, current understanding of the mechanisms of synthesis and regulation of extracellular polysaccharides is yet to fully explain their structural intricacy and functional variability.

Stable expression of silencing-suppressor protein enhances the performance and longevity of an engineered metabolic pathway

Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP-AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application.

A promoter-level mammalian expression atlas

Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

CD1 expression and CD1-restricted T cell activity in normal and tumour-bearing human liver

CD1d-restricted natural killer T (NKT) cells expressing invariant Valpha14Jalpha18 T cell receptor alpha-chains are abundant in murine liver and are implicated in the control of malignancy, infection and autoimmunity. Invariant NKT cells have potent anti-metastatic effects in mice and phase I clinical trials involving their homologues in humans are ongoing. However, invariant NKT cells are less abundant in human liver ( approximately 0.5% of hepatic T cells) than in murine liver (up to 50%) and it is not known if other hepatic T cells are CD1-restricted. We have examined expression of CD1a, CD1b, CD1c and CD1d mRNA and protein in human liver and evaluated the reactivity of mononuclear cells (MNC) from histologically normal and tumour-bearing human liver specimens against these CD1 isoforms. Messenger RNA for all CD1 isotypes was detectable in all liver samples. CD1c and CD1d were expressed at the protein level by hepatic MNC. CD1d, only, was detectable at the cell surface, but CD1c and CD1d were found at an intracellular location in significant numbers of liver MNC. CD1b was not expressed by MNC from healthy livers but was detectable within MNC in all tumour samples tested. Hepatic T cells exhibited reactivity against C1R cells expressing transfected CD1c and CD1d, but neither CD1a nor CD1b. These cells secreted interferon-gamma (IFN-gamma) but not interleukin-4 (IL-4) upon stimulation. In contrast, similar numbers of peripheral T cells released 13- and 16-fold less IFN-gamma in response to CD1c and CD1d, respectively. CD1c and CD1d expression and T cell reactivity were not altered in tumour-bearing liver specimens compared to histologically normal livers. These data suggest that, in addition to invariant CD1d-restricted NKT cells, autoreactive T cells that recognise CD1c and CD1d and release inflammatory cytokines are abundant in human liver.