Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays. We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout. Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.Burroughs Wellcome Fund (Career Award at the Scientific Interface

Designing a road traffic model for the cross-sectoral analysis of future national infrastructure

This paper presents a UK national road traffic model developed as part of the ITRC MISTRAL - a large interdisciplinary project of the Infrastructure Transitions Research Consortium (ITRC). The proposed model includes passenger and freight vehicle flows on major UK roads and predicts future demand in the form of an inter-zonal origin-destination matrix, using and elasticity-based simulation approach. An important part of the model is the network assignment step during which predicted flows are assigned to the road network. This allows for the assessment of road capacity utilisation and facilitates the identification of "pinch points" where future infrastructure investments might be targeted. Several policy interventions are studied in the paper, including road expansion with additional lanes, new road development and vehicle electrification. The model also explicitly considers cross-sectoral interdependencies with other infrastructure networks, primarily with the energy sector where the transport sector is the largest consumer, the digital communications sector, water supply and waste management. In future extensions, the model will also be able to estimate the environmental footprint and assess the risk and resilience of the transport network. This model has the potential to inform policy makers about the long-term performance of UK road infrastructure, considering a range of possible future scenarios for population growth, technological innovation and climate change

The implications of ambitious decarbonisation of heat and road transport for Britain’s net zero carbon energy systems

Decarbonisation of heating and road transport are regarded as necessary but very challenging steps on the pathway to net zero carbon emissions. Assessing the most efficient routes to decarbonise these sectors requires an integrated view of energy and road transport systems. Here we describe how a national gas and electricity transmission network model was extended to represent multiple local energy systems and coupled with a national energy demand and road transport model. The integrated models were applied to assess a range of technologies and policies for heating and transport where the UK’s 2050 net zero carbon emissions target is met. Overall, annual primary energy use is projected to reduce by between 25% and 50% by 2050 compared to 2015, due to ambitious efficiency improvements within homes and vehicles. However, both annual and peak electricity demands in 2050 are more than double compared with 2015. Managed electric vehicle charging could save 14TWh/year in gas-fired power generation at peak times, and associated emissions, whilst vehicle-to-grid services could provide 10GW of electricity supply during peak hours. Together, managed vehicle charging, and vehicle-to-grid supplies could result in a 16% reduction in total annual energy costs. The provision of fast public charging facilities could reduce peak electricity demand by 17GW and save an estimated £650 million annually. Although using hydrogen for heating and transport spreads the hydrogen network costs between homeowners and motorists, it is still estimated to be more costly overall compared to an all-electric scenario. Bio-energy electricity generation plants with carbon capture and storage are required to drive overall energy system emissions to net zero, utilisation of which is lowest when heating is electrified, and road transport consists of a mix of electric and hydrogen fuel-cell vehicles. The analysis demonstrates the need for an integrated systems approach to energy and transport policies and for coordination between national and local governments

Digital MDA for enumeration of total nucleic acid contamination

Multiple displacement amplification (MDA) is an isothermal, sequence-independent method for the amplification of high molecular weight DNA that is driven by ϕ29 DNA polymerase (DNAP). Here we report digital MDA (dMDA), an ultrasensitive method for quantifying nucleic acid fragments of unknown sequence. We use the new assay to show that our custom ϕ29 DNAP preparation is free of contamination at the limit of detection of the dMDA assay (1 contaminating molecule per assay microliter). Contamination in commercially available preparations is also investigated. The results of the dMDA assay provide strong evidence that the so-called ‘template-independent’ MDA background can be attributed to high-molecular weight contaminants and is not primer-derived in the commercial kits tested. dMDA is orders of magnitude more sensitive than PCR-based techniques for detection of microbial genomic DNA fragments and opens up new possibilities for the ultrasensitive quantification of DNA fragments in a wide variety of application areas using MDA chemistry and off-the-shelf hardware developed for digital PCR

Quantitative evaluation of the cracking of carbon anodes by image analysis for aluminum industry

Quality of carbon anodes used in aluminum industry affects significantly the electrolytic cell performance including the energy consumption. One of the important factors that influence the anode quality is the cracking of anodes. Quantitative evaluation of this problem helps identify the causes and consequently allows the necessary corrective actions to be taken. The characterization of the baked anode cracking was done by determining the sizes (width, length) of the cracks and their distribution on the anode surface. This work was carried out using the image analysis technique. Two types of cracking, horizontal and vertical, were investigated for industrial baked anodes. The results show the types of cracks encountered in industrial anodes and indicate the regions susceptible to the formation of cracks. In this article, the characterization method will be described, and the results on the anode crack formation will be presented

Single-cell and metagenomic analyses indicate a fermentative and saccharolytic lifestyle for members of the OP9 lineage

OP9 is a yet-uncultivated bacterial lineage found in geothermal systems, petroleum reservoirs, anaerobic digesters and wastewater treatment facilities. Here we use single-cell and metagenome sequencing to obtain two distinct, nearly complete OP9 genomes, one constructed from single cells sorted from hot spring sediments and the other derived from binned metagenomic contigs from an in situ-enriched cellulolytic, thermophilic community. Phylogenomic analyses support the designation of OP9 as a candidate phylum for which we propose the name ‘Atribacteria’. Although a plurality of predicted proteins is most similar to those from Firmicutes, the presence of key genes suggests a diderm cell envelope. Metabolic reconstruction from the core genome suggests an anaerobic lifestyle based on sugar fermentation by Embden–Meyerhof glycolysis with production of hydrogen, acetate and ethanol. Putative glycohydrolases and an endoglucanase may enable catabolism of (hemi)cellulose in thermal environments. This study lays a foundation for understanding the physiology and ecological role of the ‘Atribacteria’.United States. National Aeronautics and Space Administration (Exobiology Grant EXO-NNX11AR78G)National Science Foundation (U.S.) (Grant MCB 0546865)National Science Foundation (U.S.) (Grant OISE 0968421)United States. Dept. of Energy (Grant DE-EE-0000716)Nevada Renewable Energy ConsortiumUnited States. Dept. of Energy. Office of Science. Joint Genome Institute (Contract DE-AC02-05CH11231

Decontamination of MDA Reagents for Single Cell Whole Genome Amplification

Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells

Different paths to the modern state in Europe: the interaction between domestic political economy and interstate competition

Theoretical work on state formation and capacity has focused mostly on early modern Europe and on the experience of western European states during this period. While a number of European states monopolized domestic tax collection and achieved gains in state capacity during the early modern era, for others revenues stagnated or even declined, and these variations motivated alternative hypotheses for determinants of fiscal and state capacity. In this study we test the basic hypotheses in the existing literature making use of the large date set we have compiled for all of the leading states across the continent. We find strong empirical support for two prevailing threads in the literature, arguing respectively that interstate wars and changes in economic structure towards an urbanized economy had positive fiscal impact. Regarding the main point of contention in the theoretical literature, whether it was representative or authoritarian political regimes that facilitated the gains in fiscal capacity, we do not find conclusive evidence that one performed better than the other. Instead, the empirical evidence we have gathered lends supports to the hypothesis that when under pressure of war, the fiscal performance of representative regimes was better in the more urbanized-commercial economies and the fiscal performance of authoritarian regimes was better in rural-agrarian economie