PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis.

Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tThis article contains supporting information online at www.pnas.org/cgi/content/full/0709632105/DC1. Freely available online through the PNAS open access option. © 2008 by The National Academy of Sciences of the USADNA-dependent RNA polymerase (Pol)IV in Arabidopsis exists in two isoforms (PolIVa and PolIVb), with NRPD1a and NRPD1b as their respective largest subunits. Both isoforms are implicated in production and activity of siRNAs and in RNA-directed DNA methylation (RdDM). Deep sequence analysis of siRNAs in WT Arabidopsis flowers and in nrpd1a and nrpd1b mutants identified >4,200 loci producing siRNAs in a PolIV-dependent manner, with PolIVb reinforcing siRNA production by PolIVa. Transposable element identity and pericentromeric localization are both features that predispose a locus for siRNA production via PolIV proteins and determine the extent to which siRNA production relies on PolIVb. Detailed analysis of DNA methylation at PolIV-dependent loci revealed unexpected deviations from the previously noted association of PolIVb-dependent siRNA production and RdDM. Notably, PolIVb functions independently in DNA methylation and siRNA generation. Additionally, we have uncovered siRNA-directed loss of DNA methylation, a process requiring both PolIV isoforms. From these findings, we infer that the role of PolIVb in siRNA production is secondary to a role in chromatin modification and is influenced by chromatin context

Transcriptomic Profiling of In Vitro Tumor-Stromal Cell Paracrine Crosstalk Identifies Involvement of the Integrin Signaling Pathway in the Pathogenesis of Mesenteric Fibrosis in Human Small Intestinal Neuroendocrine Neoplasms.

Aim: Analysis of the pathophysiology of mesenteric fibrosis (MF) in small intestinal neuroendocrine tumors (SI-NETs) in an in vitro paracrine model and in human SI-NET tissue samples. Methods: An indirect co-culture model of SI-NET cells KRJ-I and P-STS with stromal cells HEK293 was designed to evaluate the paracrine effects on cell metabolic activity, gene expression by RT2 PCR Profilers to analyse cancer and fibrosis related genes, and RNA sequencing. The integrin signaling pathway, a specific Ingenuity enriched pathway, was further explored in a cohort of human SI-NET tissues by performing protein analysis and immunohistochemistry. Results: RT Profiler array analysis demonstrated several genes to be significantly up- or down-regulated in a cell specific manner as a result of the paracrine effect. This was further confirmed by employing RNA sequencing revealing multiple signaling pathways involved in carcinogenesis and fibrogenesis that were significantly affected in these cell lines. A significant upregulation in the expression of various integrin pathway - related genes was identified in the mesenteric mass of fibrotic SI-NET as confirmed by RT-qPCR and immunohistochemistry. Protein analysis demonstrated downstream activation of the MAPK and mTOR pathways in some patients with fibrotic SI-NETs. Conclusion: This study has provided the first comprehensive analysis of the crosstalk of SI-NET cells with stromal cells. A novel pathway - the integrin pathway - was identified and further validated and confirmed in a cohort of human SI-NET tissue featured by a dual role in fibrogenesis/carcinogenesis within the neoplastic fibrotic microenvironment

An enhanced toolkit for the generation of knockout and marker-free fluorescent Plasmodium chabaudi.

The rodent parasite Plasmodium chabaudi is an important in vivo model of malaria. The ability to produce chronic infections makes it particularly useful for investigating the development of anti- Plasmodium immunity, as well as features associated with parasite virulence during both the acute and chronic phases of infection. P. chabaudi also undergoes asexual maturation (schizogony) and erythrocyte invasion in culture, so offers an experimentally-amenable in vivo to in vitro model for studying gene function and drug activity during parasite replication. To extend the usefulness of this model, we have further optimised transfection protocols and plasmids for P. chabaudi and generated stable, fluorescent lines that are free from drug-selectable marker genes. These mother-lines show the same infection dynamics as wild-type parasites throughout the lifecycle in mice and mosquitoes; furthermore, their virulence can be increased by serial blood passage and reset by mosquito transmission. We have also adapted the large-insert, linear PlasmoGEM vectors that have revolutionised the scale of experimental genetics in another rodent malaria parasite and used these to generate barcoded P. chabaudi gene-deletion and -tagging vectors for transfection in our fluorescent P. chabaudi mother-lines. This produces a tool-kit of P. chabaudi lines, vectors and transfection approaches that will be of broad utility to the research community

The antimalarial efficacy and mechanism of resistance of the novel chemotype DDD01034957.

New antimalarial therapeutics are needed to ensure that malaria cases continue to be driven down, as both emerging parasite resistance to frontline chemotherapies and mosquito resistance to current insecticides threaten control programmes. Plasmodium, the apicomplexan parasite responsible for malaria, causes disease pathology through repeated cycles of invasion and replication within host erythrocytes (the asexual cycle). Antimalarial drugs primarily target this cycle, seeking to reduce parasite burden within the host as fast as possible and to supress recrudescence for as long as possible. Intense phenotypic drug screening efforts have identified a number of promising new antimalarial molecules. Particularly important is the identification of compounds with new modes of action within the parasite to combat existing drug resistance and suitable for formulation of efficacious combination therapies. Here we detail the antimalarial properties of DDD01034957-a novel antimalarial molecule which is fast-acting and potent against drug resistant strains in vitro, shows activity in vivo, and possesses a resistance mechanism linked to the membrane transporter PfABCI3. These data support further medicinal chemistry lead-optimization of DDD01034957 as a novel antimalarial chemical class and provide new insights to further reduce in vivo metabolic clearance

The antimalarial efficacy and mechanism of resistance of the novel chemotype DDD01034957.

New antimalarial therapeutics are needed to ensure that malaria cases continue to be driven down, as both emerging parasite resistance to frontline chemotherapies and mosquito resistance to current insecticides threaten control programmes. Plasmodium, the apicomplexan parasite responsible for malaria, causes disease pathology through repeated cycles of invasion and replication within host erythrocytes (the asexual cycle). Antimalarial drugs primarily target this cycle, seeking to reduce parasite burden within the host as fast as possible and to supress recrudescence for as long as possible. Intense phenotypic drug screening efforts have identified a number of promising new antimalarial molecules. Particularly important is the identification of compounds with new modes of action within the parasite to combat existing drug resistance and suitable for formulation of efficacious combination therapies. Here we detail the antimalarial properties of DDD01034957-a novel antimalarial molecule which is fast-acting and potent against drug resistant strains in vitro, shows activity in vivo, and possesses a resistance mechanism linked to the membrane transporter PfABCI3. These data support further medicinal chemistry lead-optimization of DDD01034957 as a novel antimalarial chemical class and provide new insights to further reduce in vivo metabolic clearance

Fatigue life of machined components

A correlation between machining process and fatigue strength of machined components clearly exists. However, a complete picture of the knowledge on this is not readily available for practical applications. This study addresses this issue by investigating the effects of machining methods on fatigue life of commonly used materials, such as titanium alloys, steel, aluminium alloys and nickel alloys from previous literature. Effects of turning, milling, grinding and different non-conventional machining processes on fatigue strength of above-mentioned materials have been investigated in detail with correlated information. It is found that the effect of materials is not significant except steel in which phase change causes volume expansion, resulting in compressive/tensile residual stresses based on the amounts of white layers. It is very complex to identify the influence of surface roughness on the fatigue strength of machined components in the presence of residual stresses. The polishing process improves the surface roughness, but removes the surface layers that contain compressive residual stresses to decrease the fatigue strength of polished specimens. The compressive and tensile residual stresses improve and reduce fatigue strength, respectively. Grinding process induces tensile residual stresses on the machined surfaces due to high temperature generation. On the other hand, milling and turning processes induce compressive residual stresses. High temperature non-conventional machining generates a network of micro-cracks on the surfaces in addition to tensile residual stresses to subsequently reduce fatigue strength of machined components. Embedded grits of abrasive water jet machining degrade the fatigue performance of components machined by this method

The dynamics and efficacy of antiviral RNA silencing: A model study

<p>Abstract</p> <p>Background</p> <p>Mathematical modeling is important to provide insight in the complicated pathway of RNA silencing. RNA silencing is an RNA based mechanism that is widely used by eukaryotes to fight viruses, and to control gene expression.</p> <p>Results</p> <p>We here present the first mathematical model that combines viral growth with RNA silencing. The model involves a plus-strand RNA virus that replicates through a double-strand RNA intermediate. The model of the RNA silencing pathway consists of cleavage of viral RNA into siRNA by Dicer, target cleavage of viral RNA via the RISC complex, and a secondary response. We found that, depending on the strength of the silencing response, different viral growth patterns can occur. Silencing can decrease viral growth, cause oscillations, or clear the virus completely. Our model can explain various observed phenomena, even when they seem contradictory at first: the diverse responses to the removal of RNA dependent RNA polymerase; different viral growth curves; and the great diversity in observed siRNA ratios.</p> <p>Conclusion</p> <p>The model presented here is an important step in the understanding of the natural functioning of RNA silencing in viral infections.</p

Earth observation for sustainable urban planning in developing countries: needs, trends, and future directions

Abstract: Cities are constantly changing and authorities face immense challenges in obtaining accurate and timely data to effectively manage urban areas. This is particularly problematic in the developing world where municipal records are often unavailable or not updated. Spaceborne earth observation (EO) has great potential for providing up-to-date spatial information about urban areas. This article reviews the application of EO for supporting urban planning. In particular, the article overviews case studies where EO was used to derive products and indicators required by urban planners. The review concludes that EO has sufficiently matured in recent years but that a shift from the current focus on purely science-driven EO applications to the provision of useful information for day-to-day decision-making and urban sustainability monitoring is clearly needed

Involvement of RDR6 in short-range intercellular RNA silencing in Nicotiana benthamiana

In plants, non-cell autonomous RNA silencing spreads between cells and over long distances. Recent work has revealed insight on the genetic and molecular components essential for cell-to-cell movement of RNA silencing in Arabidopsis. Using a local RNA silencing assay, we report on a distinct mechanism that may govern the short-range (6–10 cell) trafficking of virus-induced RNA silencing from epidermal to neighbouring palisade and spongy parenchyma cells in Nicotiana benthamiana. This process involves a previously unrecognised function of the RNA-dependent RNA polymerase 6 (RDR6) gene. Our data suggest that plants may have evolved distinct genetic controls in intercellular RNA silencing among different types of cells