Bead-Droplet Reactor for High-Fidelity Solid-Phase Enzymatic DNA Synthesis

Solid-phase synthesis techniques underpin the synthesis of DNA, oligopeptides, oligosaccharides, and combinatorial libraries for drug discovery. State-of-the-art solid-phase synthesizers can produce oligonucleotides up to 200-300 nucleotides while using excess reagents. Accumulated errors over multiple reaction cycles prevent the synthesis of longer oligonucleotides for the genome scale engineering of synthetic biological systems. The sources of these errors in synthesis columns remains poorly understood. Here we show that bead-bead stacking significantly contributes to reaction errors in columns by analyzing enzymatic coupling of fluorescently labelled nucleotides onto the initiated beads along with porosity, particle tracking and diffusion calculations. To circumvent stacking, we introduce dielectrophoretic bead-droplet reactor (DBDR); a novel approach to synthesize on individual microbeads within microdroplets. Dielectrophoretic force overcomes the droplet-medium interfacial tension to encapsulate and eject individual beads from microdroplets in a droplet microfluidic device. Faster reagent diffusion in droplets, and non-uniform electric field induced enhancement in reagent concentration at its surface can improve reaction fidelities in DBDR. Fluorescence comparisons suggest around 3-fold enhancement of reaction fidelity compared to columns. DBDR can potentially enable the high-purity synthesis of arbitrarily long strands of DNA to meet the emerging demands in healthcare, environment, agriculture, materials, and computing

Albumin-Associated Lipids Regulate Human Embryonic Stem Cell Self-Renewal

BACKGROUND: Although human embryonic stem cells (hESCs) hold great promise as a source of differentiated cells to treat several human diseases, many obstacles still need to be surmounted before this can become a reality. First among these, a robust chemically-defined system to expand hESCs in culture is still unavailable despite recent advances in the understanding of factors controlling hESC self-renewal. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we attempted to find new molecules that stimulate long term hESC self-renewal. In order to do this, we started from the observation that a commercially available serum replacement product has a strong positive effect on the expansion of undifferentiated hESCs when added to a previously reported chemically-defined medium. Subsequent experiments demonstrated that the active ingredient within the serum replacement is lipid-rich albumin. Furthermore, we show that this activity is trypsin-resistant, strongly suggesting that lipids and not albumin are responsible for the effect. Consistent with this, lipid-poor albumin shows no detectable activity. Finally, we identified the major lipids bound to the lipid-rich albumin and tested several lipid candidates for the effect. CONCLUSIONS/SIGNIFICANCE: Our discovery of the role played by albumin-associated lipids in stimulating hESC self-renewal constitutes a significant advance in the knowledge of how hESC pluripotency is maintained by extracellular factors and has important applications in the development of increasingly chemically defined hESC culture systems

Floods: vulnerability, risks and management. A joint report of ETC CCA and ICM

This report describes floods in a European context with the purpose of highlighting factors that contribute to the occurrence and adverse consequences of floods, and possibilities to reduce flood risks from inland waters and rainfall. It includes a discussion on changes in flood patterns and illustrates how different scenarios for climate change may affect vulnerability to floods and flood risks. The report provides illustrative examples of flood risk management from the local to European level

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications.

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression

The availability of novelty sweets within the high school fringe

Background Reducing sugar consumption is a primary focus of current global public health policy. Achieving 5% of total energy from free sugars will be difficult acknowledging the concentration of free sugars in sugar sweetened beverages, confectionery and as hidden sugars in many savoury items. The expansion of the novelty sweet market in the UK has significant implications for children and young adults as they contribute to dental caries, dental erosion and obesity. Objective To identify the most available types of novelty sweets within the high school fringe in Cardiff, UK and to assess their price range and where and how they were displayed in shops. Subjects and methods Shops within a ten minute walking distance around five purposively selected high schools in the Cardiff aea representing different levels of deprivation were visited. Shops in Cardiff city centre and three supermarkets were also visited to identify the most commonly available novelty sweets. Results The ten most popular novelty sweets identified in these scoping visits were (in descending order): Brain Licker, Push Pop, Juicy Drop, Lickedy Lips, Big Baby Pop, Vimto candy spray, Toxic Waste, Tango candy spray, Brain Blasterz Bitz and Mega Mouth candy spray. Novelty sweets were located on low shelves which were accessible to all age-groups in 73% (14 out of 19) of the shops. Novelty sweets were displayed in the checkout area in 37% (seven out of 19) shops. The price of the top ten novelty sweets ranged from 39p to £1. Conclusion A wide range of acidic and sugary novelty sweets were easily accessible and priced within pocket money range. Those personnel involved in delivering dental and wider health education or health promotion need to be aware of recent developments in children's confectionery. The potential effects of these novelty sweets on both general and dental health require further investigation

First Observation of τ→3πηντ\tau\to 3\pi\eta\nu_{\tau} and τ→f1πντ\tau\to f_{1}\pi\nu_{\tau} Decays

We have observed new channels for $\tau$ decays with an $\eta$ in the final state. We study 3-prong tau decays, using the $\eta\to\gamma\gamma$ and \eta\to 3\piz decay modes and 1-prong decays with two \piz's using the $\eta\to\gamma\gamma$ channel. The measured branching fractions are \B(\tau^{-}\to \pi^{-}\pi^{-}\pi^{+}\eta\nu_{\tau}) =(3.4^{+0.6}_{-0.5}\pm0.6)\times10^{-4} and \B(\tau^{-}\to \pi^{-}2\piz\eta\nu_{\tau} =(1.4\pm0.6\pm0.3)\times10^{-4}. We observe clear evidence for $f_1\to\eta\pi\pi$ substructure and measure \B(\tau^{-}\to f_1\pi^{-}\nu_{\tau})=(5.8^{+1.4}_{-1.3}\pm1.8)\times10^{-4}. We have also searched for $\eta'(958)$ production and obtain 90% CL upper limits \B(\tau^{-}\to \pi^{-}\eta'\nu_\tau)<7.4\times10^{-5} and \B(\tau^{-}\to \pi^{-}\piz\eta'\nu_\tau)<8.0\times10^{-5}.Comment: 11 page postscript file, postscript file also available through http://w4.lns.cornell.edu/public/CLN