“Paper Machine” for Molecular Diagnostics

Clinical tests based on primer-initiated amplification of specific nucleic acid sequences achieve high levels of sensitivity and specificity. Despite these desirable characteristics, these tests have not reached their full potential because their complexity and expense limit their usefulness to centralized laboratories. This paper describes a device that integrates sample preparation and loop-mediated isothermal amplification (LAMP) with end point detection using a hand-held UV source and camera phone. The prototype device integrates paper microfluidics (to enable fluid handling) and a multilayer structure, or a “paper machine”, that allows a central patterned paper strip to slide in and out of fluidic path and thus allows introduction of sample, wash buffers, amplification master mix, and detection reagents with minimal pipetting, in a hand-held, disposable device intended for point-of-care use in resource-limited environments. This device creates a dynamic seal that prevents evaporation during incubation at 65 °C for 1 h. This interval is sufficient to allow a LAMP reaction for the Escherichia coli malB gene to proceed with an analytical sensitivity of 1 double-stranded DNA target copy. Starting with human plasma spiked with whole, live E. coli cells, this paper demonstrates full integration of sample preparation with LAMP amplification and end point detection with a limit of detection of 5 cells. Further, it shows that the method used to prepare sample enables concentration of DNA from sample volumes commonly available from fingerstick blood draw.Chemistry and Chemical Biolog

High-throughput comparison of gene fitness among related bacteria

BACKGROUND: The contribution of a gene to the fitness of a bacterium can be assayed by whether and to what degree the bacterium tolerates transposon insertions in that gene. We use this fact to compare the fitness of syntenic homologous genes among related Salmonella strains and thereby reveal differences not apparent at the gene sequence level. RESULTS: A transposon Tn5 derivative was used to construct mutants in Salmonella Typhimurium ATCC14028 (STM1) and Salmonella Typhi Ty2 (STY1), which were then grown in rich media. The locations of 234,152 and 53,556 integration sites, respectively, were mapped by sequencing. These data were compared to similar data available for a different Ty2 isolate (STY2) and essential genes identified in E. coli K-12 (ECO). Of 277 genes considered essential in ECO, all had syntenic homologs in STM1, STY1, and STY2, and all but nine genes were either devoid of transposon insertions or had very few. For three of these nine genes, part of the annotated gene lacked transposon integrations (yejM, ftsN and murB). At least one of the other six genes, trpS, had a potentially functionally redundant gene encoded elsewhere in Salmonella but not in ECO. An additional 165 genes were almost entirely devoid of transposon integrations in all three Salmonella strains examined, including many genes associated with protein and DNA synthesis. Four of these genes (STM14_1498, STM14_2872, STM14_3360, and STM14_5442) are not found in E. coli. Notable differences in the extent of gene selection were also observed among the three different Salmonella isolates. Mutations in hns, for example, were selected against in STM1 but not in the two STY strains, which have a defect in rpoS rendering hns nonessential. CONCLUSIONS: Comparisons among transposon integration profiles from different members of a species and among related species, all grown in similar conditions, identify differences in gene contributions to fitness among syntenic homologs. Further differences in fitness profiles among shared genes can be expected in other selective environments, with potential relevance for comparative systems biology

Comparison of Motion Correction Methods Incorporating Motion Modelling for PET/CT Using a Single Breath Hold Attenuation Map

Introducing motion models into respiratory motion correction methods can lead to a reduction in blurring and artefacts. However, the pool of research where motion modelling methods are applied to combined positron emission tomography and computed tomography is relatively shallow. Previous work used non-attenuation corrected time-of-flight data to fit motion models, not only to motion correct the volumes themselves, but also to warp a single attenuation map to the positions of the initial gated data. This work seeks to extend previous work to offer a comparison of respiratory motion correction methods, not only with and without motion models, but also to compare pair-wise and group-wise registration techniques, on simulation data, in a low count scenario, where the attenuation map is from a pseudo-breath hold acquisition. To test the methods, 4-Dimensional Extended Cardiac Torso images are constructed, simulated and reconstructed without attenuation correction, then motion corrected using one of pair-wise, pair-wise with motion model, group-wise and group-wise with motion model registration. Next these motion corrected volumes are registered to the breath hold attenuation map. The positron emission tomography data are then reconstructed using deformed attenuation maps and motion corrected. Evaluation compares the results of these methods against non-motion corrected and motion free examples. Results indicate that the incorporation of motion models and group-wise registration, improves contrast and quantification

Scope for improved eco-efficiency varies among diverse cropping systems

Global food security requires eco-efficient agriculture to produce the required food and fiber products concomitant with ecologically efficient use of resources. This eco-efficiency concept is used to diagnose the state of agricultural production in China (irrigated wheat–maize double-cropping systems), Zimbabwe (rainfed maize systems), and Australia (rainfed wheat systems). More than 3,000 surveyed crop yields in these three countries were compared against simulated grain yields at farmer-specified levels of nitrogen (N) input. Many Australian commercial wheat farmers are both close to existing production frontiers and gain little prospective return from increasing their N input. Significant losses of N from their systems, either as nitrous oxide emissions or as nitrate leached from the soil profile, are infrequent and at low intensities relative to their level of grain production. These Australian farmers operate close to eco-efficient frontiers in regard to N, and so innovations in technologies and practices are essential to increasing their production without added economic or environmental risks. In contrast, many Chinese farmers can reduce N input without sacrificing production through more efficient use of their fertilizer input. In fact, there are real prospects for the double-cropping systems on the North China Plain to achieve both production increases and reduced environmental risks. Zimbabwean farmers have the opportunity for significant production increases by both improving their technical efficiency and increasing their level of input; however, doing so will require improved management expertise and greater access to institutional support for addressing the higher risks. This paper shows that pathways for achieving improved eco-efficiency will differ among diverse cropping systems