Efficient in vitro RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, Brugia malayi

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode <it>Caenorhabditis elegans</it>, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from <it>C. elegans</it>.</p> <p>Results</p> <p>We report improved and effective <it>in vitro </it>RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, <it>Brugia malayi</it>. The cellular disorganization observed in <it>B. malayi </it>embryos following RNAi targeting the genes encoding γ-tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their <it>C. elegans </it>orthologs. Targeting the <it>B. malayi </it>cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in <it>C. elegans</it>. Cellular phenotypes induced by our <it>in vitro </it>RNAi procedure can be observed by immunofluorescence in as little as one week.</p> <p>Conclusions</p> <p>We observed cytological defects following RNAi targeting all seven <it>B. malayi </it>transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism <it>C. elegans</it>. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis.</p

Two Birds with One Stone? Possible Dual-Targeting H1N1 Inhibitors from Traditional Chinese Medicine

The H1N1 influenza pandemic of 2009 has claimed over 18,000 lives. During this pandemic, development of drug resistance further complicated efforts to control and treat the widespread illness. This research utilizes traditional Chinese medicine Database@Taiwan (TCM Database@Taiwan) to screen for compounds that simultaneously target H1 and N1 to overcome current difficulties with virus mutations. The top three candidates were de novo derivatives of xylopine and rosmaricine. Bioactivity of the de novo derivatives against N1 were validated by multiple machine learning prediction models. Ability of the de novo compounds to maintain CoMFA/CoMSIA contour and form key interactions implied bioactivity within H1 as well. Addition of a pyridinium fragment was critical to form stable interactions in H1 and N1 as supported by molecular dynamics (MD) simulation. Results from MD, hydrophobic interactions, and torsion angles are consistent and support the findings of docking. Multiple anchors and lack of binding to residues prone to mutation suggest that the TCM de novo derivatives may be resistant to drug resistance and are advantageous over conventional H1N1 treatments such as oseltamivir. These results suggest that the TCM de novo derivatives may be suitable candidates of dual-targeting drugs for influenza.National Science Council of Taiwan (NSC 99-2221-E-039-013-)Committee on Chinese Medicine and Pharmacy (CCMP100-RD-030)China Medical University and Asia University (CMU98-TCM)China Medical University and Asia University (CMU99-TCM)China Medical University and Asia University (CMU99-S-02)China Medical University and Asia University (CMU99-ASIA-25)China Medical University and Asia University (CMU99-ASIA-26)China Medical University and Asia University (CMU99-ASIA-27)China Medical University and Asia University (CMU99-ASIA-28)Taiwan Department of Health. Clinical Trial and Research Center of Excellence (DOH100-TD-B-111-004)Taiwan Department of Health. Cancer Research Center of Excellence (DOH100-TD-C-111-005

Decadal slowdown of a land-terminating sector of the Greenland Ice Sheet despite warming

Ice flow along land-terminating margins of the Greenland Ice Sheet (GIS) varies considerably in response to fluctuating inputs of surface meltwater to the bed of the ice sheet. Such inputs lubricate the ice-bed interface, transiently speeding up the flow of ice. Greater melting results in faster ice motion during summer, but slower motion over the subsequent winter, owing to the evolution of an efficient drainage system that enables water to drain from regions of the ice-sheet bed that have a high basal water pressure. However, the impact of hydrodynamic coupling on ice motion over decadal timescales remains poorly constrained. Here we show that annual ice motion across an 8,000-km2 land-terminating region of the west GIS margin, extending to 1,100 m above sea level, was 12 slower in 2007-14 compared with 1985-94, despite a 50 increase in surface meltwater production. Our findings suggest that, over these three decades, hydrodynamic coupling in this section of the ablation zone resulted in a net slowdown of ice motion (not a speed-up, as previously postulated). Increases in meltwater production from projected climate warming may therefore further reduce the motion of land-terminating margins of the GIS. Our findings suggest that these sectors of the ice sheet are more resilient to the dynamic impacts of enhanced meltwater production than previously thought. © 2015 Macmillan Publishers Limited. All rights reserved

Ice discharge uncertainties in Northeast Greenland from boundary conditions and climate forcing of an ice flow model

In order to understand ice sheet response to climate change, it is critical to examine errors associated with ice flow model boundary conditions and forcing. It is also important to understand how these errors propagate through numerical ice sheet models and contribute to uncertainty in model output. Using established uncertainty quantification methods within the Ice Sheet System Model (ISSM), we investigate the sensitivity of ice flow within the Northeast Greenland Ice Stream (NEGIS) to key fields, including ice viscosity and basal drag, and compare them with model sensitivity to climate forcing. In addition, we examine how errors in model input manifest as mass flux uncertainties during a forward simulation of the NEGIS from 1989 to 2010. Overall, we find that mass flux is most uncertain in the main outlets, Nioghalvfjerdsbræ and Zachariæ Isstrøm, and that mass flux is most sensitive to basal drag, though errors associated with basal drag are poorly constrained and difficult to quantify. Given our knowledge of errors associated with the thermal properties of ice, we estimate that in the ablation area, the effects of cryohydrologic warming contribute over 4 times more mass flux uncertainty that do errors in geothermal heat flux. We find that NEGIS total ice discharge is associated with a 0.7 Gt/yr (2.6%) uncertainty due to errors in geothermal heat flux and a 3.3 Gt/yr (11.6%) uncertainty due to the added effects of cryohydrologic warming. In comparison, errors in surface mass balance contribute 4.5 Gt/yr to NEGIS total discharge uncertainty. Key Points Mass flux is most sensitive to basal drag Mass flux is the most uncertain at the 79North outlet Geothermal heat flux uncertainty is less than that from cryohydrologic warming

Ice discharge uncertainties in Northeast Greenland from boundary conditions and climate forcing of an ice flow model

In order to understand ice sheet response to climate change, it is critical to examine errors associated with ice flow model boundary conditions and forcing. It is also important to understand how these errors propagate through numerical ice sheet models and contribute to uncertainty in model output. Using established uncertainty quantification methods within the Ice Sheet System Model (ISSM), we investigate the sensitivity of ice flow within the Northeast Greenland Ice Stream (NEGIS) to key fields, including ice viscosity and basal drag, and compare them with model sensitivity to climate forcing. In addition, we examine how errors in model input manifest as mass flux uncertainties during a forward simulation of the NEGIS from 1989 to 2010. Overall, we find that mass flux is most uncertain in the main outlets, Nioghalvfjerdsbræ and Zachariæ Isstrøm, and that mass flux is most sensitive to basal drag, though errors associated with basal drag are poorly constrained and difficult to quantify. Given our knowledge of errors associated with the thermal properties of ice, we estimate that in the ablation area, the effects of cryohydrologic warming contribute over 4 times more mass flux uncertainty that do errors in geothermal heat flux. We find that NEGIS total ice discharge is associated with a 0.7 Gt/yr (2.6%) uncertainty due to errors in geothermal heat flux and a 3.3 Gt/yr (11.6%) uncertainty due to the added effects of cryohydrologic warming. In comparison, errors in surface mass balance contribute 4.5 Gt/yr to NEGIS total discharge uncertainty. Key Points Mass flux is most sensitive to basal drag Mass flux is the most uncertain at the 79North outlet Geothermal heat flux uncertainty is less than that from cryohydrologic warming