RNA interference in a cestode reveals specific silencing of selected highly expressed gene transcripts

Evolving RNA interference (RNAi) platforms are providing opportunities to probe gene function in parasitic helminths using reverse genetics. Although relatively robust methods for the application of RNAi in parasitic flatworms have been established, reports of successful RNAi are confined to three genera and there are no known reports of the application of RNAi to the class Cestoda. Here we report the successful application of RNAi to a cestode. Our target species was the common ruminant tapeworm, Moniezia expansa which can significantly impact the health/productivity of cattle, sheep and goats. Initial efforts aimed to silence the neuronally expressed neuropeptide F gene (Me-npf-1), which encodes one of the most abundant neuropeptides in flatworms and a homologue of vertebrate neuropeptide Y (NPY). Double stranded (ds)RNAs, delivered by electroporation and soaking (4-8 h), failed to trigger consistent Me-npf-1 transcript knock-down in adult worms; small interfering RNAs (siRNAs) were also ineffective. Identical approaches resulted in significant and consistent transcript knock-down of actin transcript (71 +/- 4%) following soaking in Me-act-1 dsRNA. Similar successes were seen with hydrophobic lipid-binding protein (Me-lbp-1), with a dsRNA inducing significant target transcript reduction (72 +/- 5%). To confirm the validity of the observed transcript knock-downs we further investigated Me-act-1 RNAi worms for associated changes in protein levels, morphology and phenotype. Me-act-1 RNAi worms displayed significant reductions in both filamentous actin immunostaining (62 +/- 3%) and the amount of actin detected in Western blots (54 +/- 13%). Morphologically, Me-act-1 RNAi worms displayed profound tegumental disruption/blebbing. Further, muscle tension recordings from Me-act-1 RNAi worms revealed a significant reduction in both the number of worms contracting in response to praziquantel (20 +/- 12%) and in their contractile ability. These data demonstrate, to our knowledge for the first time, a functional RNAi pathway in a cestode and show that the robust knock-down of abundant gene transcripts is achievable using long dsRNAs following short exposure times. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Nitric oxide and hypoxia

NO (nitric oxide) can affect mitochondrial function by interacting with the cytochrome c oxidase (complex IV) of the electron transport chain in a manner that is reversible and in competition with oxygen. Concentrations of NO too low to inhibit respiration can trigger cell defence response mechanisms involving reactive oxygen species and various signalling molecules such as nuclear factor kappa B and AMP kinase. Inhibition of mitochondrial respiration by NO at low oxygen concentrations can cause so-called metabolic hypoxia and divert oxygen towards other oxygen-dependent systems. Such a diversion reactivates prolyl hydroxylases and thus accounts for the prevention by NO of the stabilization of hypoxia-inducible transcription factor. In certain circumstances NO interacts with superoxide radical to form peroxynitrite, which can affect the action of key enzymes, such as mitochondrial complex I, by S-nitrosation. This chapter discusses the physiological and pathophysiological implications of the interactions of NO with the cytochrome c oxidase.

Sources of Software Requirements Change from the Perspectives of Development and Maintenance

Changes to software requirements occur during initial development and subsequent to delivery, posing a risk to cost and quality while at the same time providing an opportunity to add value. Provision of a generic change source taxonomy will support requirements change risk visibility, and also facilitate richer recording of both pre- and post-delivery change data. In this paper we present a collaborative study to investigate and classify sources of requirements change, drawing comparison between those pertaining to software development and maintenance. We begin by combining evolution, maintenance and software lifecycle research to derive a definition of software maintenance, which provides the foundation for empirical context and comparison. Previously published change ‘causes’ pertaining to development are elicited from the literature, consolidated using expert knowledge and classified using card sorting. A second study incorporating causes of requirements change during software maintenance results in a taxonomy which accounts for the entire evolutionary progress of applications software. We conclude that the distinction between the terms maintenance and development is imprecise, and that changes to requirements in both scenarios arise due to a combination of factors contributing to requirements uncertainty and events that trigger change. The change trigger taxonomy constructs were initially validated using a small set of requirements change data, and deemed sufficient and practical as a means to collect common requirements change statistics across multiple projects.

Chemotherapy-Induced Activation of ADAM-17: A Novel Mechanism of Drug Resistance in Colorectal Cancer

Purpose: We have shown previously that exposure to anticancer drugs can trigger the activation of human epidermal receptor survival pathways in colorectal cancer (CRC). In this study, we examined the role of ADAMs (a disintegrin and metalloproteinases) and soluble growth factors in this acute drug resistance mechanism.

Experimental Design: In vitro and in vivo models of CRC were assessed. ADAM-17 activity was measured using a fluorometric assay. Ligand shedding was assessed by ELISA or Western blotting. Apoptosis was assessed by flow cytometry and Western blotting.

Results: Chemotherapy (5-fluorouracil) treatment resulted in acute increases in transforming growth factor-a, amphiregulin, and heregulin ligand shedding in vitro and in vivo that correlated with significantly increased ADAM-17 activity. Small interfering RNA–mediated silencing and pharmacologic inhibition confirmed that ADAM-17 was the principal ADAM involved in this prosurvival response. Furthermore, overexpression of ADAM-17 significantly decreased the effect of chemotherapy on tumor growth and apoptosis. Mechanistically, we found that ADAM-17 not only regulated phosphorylation of human epidermal receptors but also increased the activity of a number of other growth factor receptors, such as insulin-like growth factor-I receptor and vascular endothelial growth factor receptor.

Conclusions: Chemotherapy acutely activates ADAM-17, which results in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. Thus, pharmacologic inhibition of ADAM-17 in conjunction with chemotherapy may have therapeutic potential for the treatment of CRC.

A Geostatistical Investigation into Changing Permeability of Sandstones During Weathering Simulations

The ability to predict the behavior of masonry materials is crucial to conserve building stone. Natural stone, such as sandstone, is not immune from the processes of weathering in the built environment and suffers from decay by granular disintegration, contour scaling, and multiple flaking. Spatial variation of rock properties is a major contributing factor to inconsistent responses to weathering. This has implications for moisture movement and salt input and output and storage, and results in unpredictability in the decay dynamics of masonry materials. This article explores the use of variography and kriging to investigate the spatial interactions between the trigger factors of stone decay, in particular, permeability and its effect on salt penetration. Sandstone blocks were used to represent fresh building stones from a weathering perspective and gave baseline characteristics for the interpretation of subsequent deterioration and decay pathways. Simulated weathering trials involved preloading a sandstone block with salt and subjecting a separate block to 20 cycles of a weathering trial designed to simulate a temperate weathering regime. Geostatistical analysis indicated differences in the spatial variation of permeability of the fresh rock and that subjected to the weathering regimes. Spatial prediction and visualization showed differences in the spatial continuity of permeability in a horizontal and vertical direction through the preloaded block after salt weathering. Continual wetting with salt and alternate heating increased permeability in a vertical direction, enabling the ingress and movement of salt and moisture more effectively through the stone.

Mapping NGO Competency to Reduce Human Vulnerability in Post-disaster Communities: Comparing Strategies in Sri Lanka and Bangladesh

Natural hazards trigger disasters, the scale of which is largely determined by vulnerability. Developing countries suffer the most from disasters due to various conditions of vulnerability which exist and there is an opportunity after disasters to take mitigative action. NGOs implementing post-disaster rehabilitation projects must be able to address the issues causing communities to live at risk of disaster and therefore must build dynamic capacity, capabilities and competencies, enabling them to operate in unstable environments. This research is built upon a theoretical framework of dynamic competency established by combining elements of disaster management, strategic management and project management theory. A number of NGOs which have implemented reconstruction and rehabilitation projects both in Sri Lanka following the Asian Tsunami and Bangladesh following Cyclone Sidr are being investigated in great depth using a causal mapping procedure. ‘Event’ specific maps have been developed for each organization in each disaster. This data will be analysed with a view to discovering the strategies which lead to vulnerability reduction in post-disaster communities and the competencies that NGOs must possess in order to achieve favourable outcomes. It is hypothesized that by building organizational capacity, capabilities and competencies to be dynamic in nature, while focusing on a more emergent strategic approach, with emphasis on adaptive capability and innovation, NGOs will be better equipped to contribute to sustainable community development through reconstruction. We believe that through this study it will be possible to glean a new understanding of social processes that emerge within community rehabilitation projects.

Stimulation of phosphate uptake and polyphosphate accumulation by activated sludge microorganisms in response to sulfite addition

Enhanced phosphate removal from wastewaters is dependent on the synthesis and intracellular accumulation of polyphosphate by sludge microorganisms. However the role played by polyphosphate in microbial metabolism and the factors that trigger its formation remain poorly-understood. Many examples of the accumulation of the biopolymer by environmental microorganisms are documented; these include a recent report of the presence of large polyphosphate inclusions in sulfur-oxidizing marine bacteria. To investigate whether any link might exist outside the marine environment between the presence of reduced sulfur compounds and enhanced levels of microbial phosphate uptake and polyphosphate accumulation, activated sludge cultures were grown under laboratory conditions in media that contained sulfite, thiosulfate, hydrosulfite or tetrathionate. Only in the presence of sulfite was there any evidence of a stimulatory effect; in medium that contained 0.5 mM sodium sulfite some 17% more phosphate was removed by the sludge, whilst there was an almost two-fold increase in intracellular polyphosphate levels. No indications of sulfite toxicity were observed.

A molecular model for drug binding to tandem repeats of telomeric G-quadruplexes.

The extreme 3'-ends of human telomeres consist of 150–250 nucleotides of single-stranded DNA sequence together with associated proteins. Small-molecule ligands can compete with these proteins and induce a conformational change in the DNA to a four-stranded quadruplex arrangement, which is also no longer a substrate for the telomerase enzyme. The modified telomere ends provide signals to the DNA-damage-response system and trigger senescence and apoptosis. Experimental structural data are available on such quadruplex complexes comprising up to four telomeric DNA repeats, but not on longer systems that are more directly relevant to the single-stranded overhang in human cells. The present paper reports on a molecular modelling study that uses Molecular Dynamics simulation methods to build dimer and tetramer quadruplex repeats. These incorporate ligand-binding sites and are models for overhang–ligand complexes.

Novel Inhibitors of the Pseudomonas aeruginosa Virulence Factor LasB: a Potential Therapeutic Approach for the Attenuation of Virulence Mechanisms in Pseudomonal Infection

Pseudomonas elastase (LasB), a metalloprotease virulence factor, is known to play a pivotal role in pseudomonal infection. LasB is secreted at the site of infection, where it exerts a proteolytic action that spans from broad tissue destruction to subtle action on components of the host immune system. The former enhances invasiveness by liberating nutrients for continued growth, while the latter exerts an immunomodulatory effect, manipulating the normal immune response. In addition to the extracellular effects of secreted LasB, it also acts within the bacterial cell to trigger the intracellular pathway that initiates growth as a bacterial bio?lm. The key role of LasB in pseudomonal virulence makes it a potential target for the development of an inhibitor as an antimicrobial agent. The concept of inhibition of virulence is a recently established antimicrobial strategy, and such agents have been termed “second-generation” antibiotics. This approach holds promise in that it seeks to attenuate virulence processes without bactericidal action and, hence, without selection pressure for the emergence of resistant strains. A potent inhibitor of LasB,N-mercaptoacetyl-Phe-Tyr-amide (Ki 41 nM) has been developed, and its ability to block these virulence processes has been assessed. It has been demonstrated that thes compound can completely block the action of LasB on protein targets that are instrumental in bio?lm formation and immunomodulation. The novel LasB inhibitor has also been employed in bacterial-cell-based assays, to reduce the growth of pseudomonal bio?lms, and to eradicate bio?lm completely when used in combination with conventional antibiotics.

The pace of Holocene vegetation change - testing for synchronous developments

Mid to high latitude forest ecosystems have undergone several major compositional changes during the Holocene. The temporal and spatial patterns of these vegetation changes hold potential information to their causes and triggers. Here we test the hypothesis that the timing of vegetation change was synchronous on a sub-continental scale, which implies a common trigger or a step-like change in climate parameters. Pollen diagrams from selected European regions were statistically divided into assemblage zones and the temporal pattern of the zone boundaries analysed. The results show that the temporal pattern of vegetation change was significantly different from random. Times of change cluster around8.2, 4.8, 3.7, and 1.2 ka, while times of higher than average stability were found around 2.1 and 5.1 ka.Compositional changes linked to the expansion of Corylus avellana and Alnus glutinosa centre around 10.6 and 9.5 ka, respectively. A climatic trigger initiating these changes may have occurred 0.5 to 1 ka earlier, respectively. The synchronous expansion of C. avellana and A. glutinosa exemplify that dispersal is not necessarily followed by population expansion. The partly synchronous, partly random expansion of A. glutinosa in adjacent European regions exemplifies that sudden synchronous population expansions are not species specific traits but vary regionally.

Near-surface temperature cycling of stone and its implications for scales of surface deterioration

Many previous studies into internal temperature gradients within stone have assumed smooth, exponential increases and decreases in sub-surface temperatures in response, for example, to diurnal patterns of heating and cooling and these have been used to explain phenomena such as large-scale contour scaling. This high-resolution experimental study, in which a porous limestone block was subjected to alternate surface heating and cooling using an infrared lamp, demonstrates that internal temperature gradients in response to short-term environmental cycles (measured in minutes) can in fact be complex and inconsistent. Results confirm the significance of very steep temperature/stress gradients within the outer 10 mm or less of exposed stone. Below this the data indicate complex patterns of temperature reversals, the amplitudes of which are attenuated with depth and which are influenced in their intensity and location by variations in the relative duration of heating and cooling phases. It is suggested that the reversals might represent ‘interference patterns’ between incoming and outgoing thermal waves, but whatever their origin they are potentially important because they occur within the zone in which many stone decay processes, especially salt weathering, operate. These processes invariably respond to temperature and moisture fluctuations, and short-term interruptions to insolation could, for example, trigger these fluctuations on numerous occasions over a day. In particular, the reversals occur at a scale that is commensurate with decay by multiple flaking and could indicate an underlying control on this previously little-researched pattern of weathering. In the context of this publication, however, the main lesson to be learned from this study is that differing scales of behaviour require different scales of enquiry.

Altered stress stimulation of inward rectifier potassium channels in Andersen-Tawil syndrome

Inward rectifier potassium channels of the Kir2 subfamily are important determinants of the electrical activity of brain and muscle cells. Genetic mutations in Kir2.1 associate with Andersen-Tawil syndrome (ATS), a familial disorder leading to stress-triggered periodic paralysis and ventricular arrhythmia. To identify the molecular mechanisms of this stress trigger, we analyze Kir channel function and localization electrophysiologically and by time-resolved confocal microscopy. Furthermore, we employ a mathematical model of muscular membrane potential. We identify a novel corticoid signaling pathway that, when activated by glucocorticoids, leads to enrichment of Kir2 channels in the plasma membranes of mammalian cell lines and isolated cardiac and skeletal muscle cells. We further demonstrate that activation of this pathway can either partly restore (40% of cases) or further impair (20% of cases) the function of mutant ATS channels, depending on the particular Kir2.1 mutation. This means that glucocorticoid treatment might either alleviate or deteriorate symptoms of ATS depending on the patient's individual Kir2.1 genotype. Thus, our findings provide a possible explanation for the contradictory effects of glucocorticoid treatment on symptoms in patients with ATS and may open new pathways for the design of personalized medicines in ATS therapy. © FASEB.

Microflare Activity Driven By Forced Magnetic Reconnection.

High cadence, multiwavelength, optical observations of a solar active region, obtained with the Swedish Solar Telescope, are presented. Two magnetic bright points are seen to separate in opposite directions at a constant velocity of 2.8 km s(-1). After a separation distance of approximate to 4400 km is reached, multiple Ellerman bombs are observed in both Ha and Ca-K images. As a result of the Ellerman bombs, periodic velocity perturbations in the vicinity of the magnetic neutral line, derived from simultaneous Michelson Doppler Imager data, are generated with amplitude +/-6 km s(-1) and wavelength approximate to 1000 km. The velocity oscillations are followed by an impulsive brightening visible in Ha and Ca-K, with a peak intensity enhancement of 63%. We interpret these velocity perturbations as the magnetic field deformation necessary to trigger forced reconnection. A time delay of approximate to 3 minutes between the Ha-wing and Ca-K observations indicates that the observed magnetic reconnection occurs at a height of similar to 200 km above the solar surface. These observations are consistent with theoretical predictions and provide the first observational evidence of microflare activity driven by forced magnetic reconnection.

Deletion of Gremlin1 increases cell proliferation and migration responses in mouse embryonic fibroblasts

Gremlin1 (Grem1) is an antagonist of bone morphogenetic proteins (BMPs) that plays a critical role in embryonic and postnatal development. Grem1 has been implicated as both a promoter and an inhibitor of cell proliferation driven by BMP-4 and other mitogens in a diverse range of cell types. Recent data showed that Grem1 can trigger angiogenesis via vascular endothelial growth factor receptor (VEGFR2) binding, highlighting that the precise modalities of Grem1 signalling require further elucidation.

Increasing the accuracy of Nitrogen Dioxide (NO2) pollution mapping using geographically weighted regression (GWR) and geostatistics.

Nitrogen Dioxide (NO2) is known to act as an environmental trigger for many respiratory illnesses. As a pollutant it is difficult to map accurately, as concentrations can vary greatly over small distances. In this study three geostatistical techniques were compared, producing maps of NO2 concentrations in the United Kingdom (UK). The primary data source for each technique was NO2 point data, generated from background automatic monitoring and background diffusion tubes, which are analysed by different laboratories on behalf of local councils and authorities in the UK. The techniques used were simple kriging (SK), ordinary kriging (OK) and simple kriging with a locally varying mean (SKlm). SK and OK make use of the primary variable only. SKlm differs in that it utilises additional data to inform prediction, and hence potentially reduces uncertainty. The secondary data source was Oxides of Nitrogen (NOx) derived from dispersion modelling outputs, at 1km x 1km resolution for the UK. These data were used to define the locally varying mean in SKlm, using two regression approaches: (i) global regression (GR) and (ii) geographically weighted regression (GWR). Based upon summary statistics and cross-validation prediction errors, SKlm using GWR derived local means produced the most accurate predictions. Therefore, using GWR to inform SKlm was beneficial in this study.