Developing Novel Nanoparticulated Imaging System using Luminescence Enhancement of Eu(III) and TB(III) by Single-Strand DNA Encapsulation

Non-toxic biosensors are encountering an increase in attention for use in understanding the fate of cells and as a diagnostic tool. Development and incorporation of suitable fluorophores into biological molecules is the key for monitoring proteins in vivo research. This study investigated the enhanced emission of Eu (III) and Tb (III) upon binding to the four DNA bases and their respective nucleotides, found the best ratio for effective energy transfer, and developing nanoparticles to deliver the biosensor into the cells. It is well known that Eu (III) and Tb (III) exhibit very distinctive photo-characteristics. The luminescence of these two lanthanides is weak due to low absorption cross sections. Conversely, the emission of both trivalent ions, upon irradiation, in aqueous solution, is strong when bound to complex ligand systems. The luminescent enhancement is the result of energy transfer (EnT) and the binding with single-stranded DNA, making these ions perfect candidates for luminescent probes (1). The emission lanthanides theory by G.A. Crosby establishes that the intramolecular energy transfer in a lanthanide complex is when the lowest triplet state energy level of the complex equals or lies above the resonance level of the lanthanide (2) To overcome the inherently low absorption of lanthanide ions, researchers have developed sensitizing fluorophores that upon excitation, transfer energy to the lanthanide (3) (4). One problem with luminescence in an aqueous solution is that another pathway is available for deactivation of the excited state of the lanthanide, in the form of vibrational energy transfer to water molecules (1). Early research shows that quenching of luminescence is minimized by using ligands which tended to encapsulate the lanthanide ion (1). Longer emission lifetimes and greater quantum yield intensities can be accomplished by either chelation by ligands (5)or encapsulation of the lanthanides. We ascertained the maximum enhancement for the lanthanide ions occurred through the interaction with the base guanine or its nucleotide guanosine 5’-monophosphate disodium salt. The research initially pursued the encapsulation of the lanthanide ions by single-strand oligonucleotides as a biosensor. However, an alternative delivery method based on inverse micelles and liposomes was developed and it proved to be economical and simple to encapsulate and deliver the biosensor into the cells. The creation of a double emulsion, or water-oil-water system, and the encapsulation (using palmitic acid as surfactant) of the water soluble biosensors were successful. This thesis determined the particle size achieved of 75nm, for both lanthanides had fallen into the nanoemulsions range. Their small size permits the nanoparticles to be injected intravenously(6). The in vitro toxicity of the nanoparticles, with both luminescence biosensors, was assessed by BCA assay. Results supported both luminescence nanoparticles biosensors were non toxic to human cells. Therefore, these NP’s have a potential to provide a unique detection signature as a contrast agent suitable for medical applications (7). It has been published that nanoparticles (NPs) can rapidly be transported to the liver (about 90%), then kidneys and other organs (8). After a period of time, the NPs are expelled from the human body through feces and urine, unless the size of the NPs is larger than 200 nm, in which case the NPs are retained / trapped by the liver. The particle size obtained in this research, 75nm, is a good indication that the biosensor will have a safe disposal from the body

FOOTER: a web tool for finding mammalian DNA regulatory regions using phylogenetic footprinting

FOOTER is a newly developed algorithm that analyzes homologous mammalian promoter sequences in order to identify transcriptional DNA regulatory ‘signals’. FOOTER uses prior knowledge about the binding site preferences of the transcription factors (TFs) in the form of position-specific scoring matrices (PSSMs). The PSSM models are generated from known mammalian binding sites from the TRANSFAC database. In a test set of 72 confirmed binding sites (most of them not present in TRANSFAC) of 19 TFs, it exhibited 83% sensitivity and 72% specificity. FOOTER is accessible over the web at

An open-data, agent-based model of alcohol related crime

The allocation of resources to challenge city centre violent crime traditionally relies on historical data to identify hot-spots. The usefulness of such data-driven approaches is limited when historical data is scarce or unavailable (e.g. planning of a new city) or insufficiently representative (e.g. does not account for novel events, such as Olympic Games). In some cities, crime data is not systematically accumulated at all. We present a graph-constrained agent based simulation model of alcohol-related violent crime that is capable of predicting areas of likely violent crime without requiring any historical data. The only inputs to our simulation are publicly available geographical data, which makes our method immediately applicable to a wide range of tasks, such as optimal city planning, police patrol optimisation, devising alcohol licensing policies. In experiments, we evaluate our model and demonstrate agreement of our model's predictions on where and when violence will occur with real-world violent crime data. Analyses indicate that our agent based model may be able to make a significant contribution to attempts to prevent violence through deterrence or by design

Association of violence with urban points of interest

The association between alcohol outlets and violence has long been recognised, and is commonly used to inform policing and licensing policies (such as staggered closing times and zoning). Less investigated, however, is the association between violent crime and other urban points of interest, which while associated with the city centre alcohol consumption economy, are not explicitly alcohol outlets. Here, machine learning (specifically, LASSO regression) is used to model the distribution of violent crime for the central 9 km2 of ten large UK cities. Densities of 620 different Point of Interest types (sourced from Ordnance Survey) are used as predictors, with the 10 most explanatory variables being automatically selected for each city. Cross validation is used to test generalisability of each model. Results show that the inclusion of additional point of interest types produces a more accurate model, with significant increases in performance over a baseline univariate alcohol-outlet only model. Analysis of chosen variables for city-specific models shows potential candidates for new strategies on a per-city basis, with combined-model variables showing the general trend in POI/violence association across the UK. Although alcohol outlets remain the best individual predictor of violence, other points of interest should also be considered when modelling the distribution of violence in city centres. The presented method could be used to develop targeted, city-specific initiatives that go beyond alcohol outlets and also consider other locations

PARalyzer: definition of RNA binding sites from PAR-CLIP short-read sequence data

Crosslinking and immunoprecipitation (CLIP) protocols have made it possible to identify transcriptome-wide RNA-protein interaction sites. In particular, PAR-CLIP utilizes a photoactivatable nucleoside for more efficient crosslinking. We present an approach, centered on the novel PARalyzer tool, for mapping high-confidence sites from PAR-CLIP deep-sequencing data. We show that PARalyzer delineates sites with a high signal-to-noise ratio. Motif finding identifies the sequence preferences of RNA-binding proteins, as well as seed-matches for highly expressed microRNAs when profiling Argonaute proteins. Our study describes tailored analytical methods and provides guidelines for future efforts to utilize high-throughput sequencing in RNA biology. PARalyzer is available at http://www.genome.duke.edu/labs/ohler/research/PARalyzer/

enoLOGOS: a versatile web tool for energy normalized sequence logos

enoLOGOS is a web-based tool that generates sequence logos from various input sources. Sequence logos have become a popular way to graphically represent DNA and amino acid sequence patterns from a set of aligned sequences. Each position of the alignment is represented by a column of stacked symbols with its total height reflecting the information content in this position. Currently, the available web servers are able to create logo images from a set of aligned sequences, but none of them generates weighted sequence logos directly from energy measurements or other sources. With the advent of high-throughput technologies for estimating the contact energy of different DNA sequences, tools that can create logos directly from binding affinity data are useful to researchers. enoLOGOS generates sequence logos from a variety of input data, including energy measurements, probability matrices, alignment matrices, count matrices and aligned sequences. Furthermore, enoLOGOS can represent the mutual information of different positions of the consensus sequence, a unique feature of this tool. Another web interface for our software, C2H2-enoLOGOS, generates logos for the DNA-binding preferences of the C2H2 zinc-finger transcription factor family members. enoLOGOS and C2H2-enoLOGOS are accessible over the web at

Pax3 expression enhances PDGF-B-induced brainstem gliomagenesis and characterizes a subset of brainstem glioma

High-grade Brainstem Glioma (BSG), also known as Diffuse Intrinsic Pontine Glioma (DIPG), is an incurable pediatric brain cancer. Increasing evidence supports the existence of regional differences in gliomagenesis such that BSG is considered a distinct disease from glioma of the cerebral cortex (CG). In an effort to elucidate unique characteristics of BSG, we conducted expression analysis of mouse PDGF-B-driven BSG and CG initiated in Nestin progenitor cells and identified a short list of expression changes specific to the brainstem gliomagenesis process, including abnormal upregulation of paired box 3 (Pax3). In the neonatal mouse brain, Pax3 expression marks a subset of brainstem progenitor cells, while it is absent from the cerebral cortex, mirroring its regional expression in glioma. Ectopic expression of Pax3 in normal brainstem progenitors in vitro shows that Pax3 inhibits apoptosis. Pax3-induced inhibition of apoptosis is p53-dependent, however, and in the absence of p53, Pax3 promotes proliferation of brainstem progenitors. In vivo, Pax3 enhances PDGF-B-driven gliomagenesis by shortening tumor latency and increasing tumor penetrance and grade, in a region-specific manner, while loss of Pax3 function extends survival of PDGF-B-driven;p53-deficient BSG-bearing mice by 33%. Importantly, Pax3 is regionally expressed in human glioma as well, with high PAX3 mRNA characterizing 40% of human BSG, revealing a subset of tumors that significantly associates with PDGFRA alterations, amplifications of cell cycle regulatory genes, and is exclusive of ACVR1 mutations. Collectively, these data suggest that regional Pax3 expression not only marks a novel subset of BSG but also contributes to PDGF-B-induced brainstem gliomagenesis

Plasma Cell Ontogeny Defined by Quantitative Changes in Blimp-1 Expression

Plasma cells comprise a population of terminally differentiated B cells that are dependent on the transcriptional regulator B lymphocyte–induced maturation protein 1 (Blimp-1) for their development. We have introduced a gfp reporter into the Blimp-1 locus and shown that heterozygous mice express the green fluorescent protein in all antibody-secreting cells (ASCs) in vivo and in vitro. In vitro, these cells display considerable heterogeneity in surface phenotype, immunoglobulin secretion rate, and Blimp-1 expression levels. Importantly, analysis of in vivo ASCs induced by immunization reveals a developmental pathway in which increasing levels of Blimp-1 expression define developmental stages of plasma cell differentiation that have many phenotypic and molecular correlates. Thus, maturation from transient plasmablast to long-lived ASCs in bone marrow is predicated on quantitative increases in Blimp-1 expression