RNAi dynamics in Juvenile Fasciola spp. Liver flukes reveals the persistence of gene silencing in vitro

Background: Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST).

Methodology/Principal Findings: Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200–320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.

Conclusions/Significance: In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection.

Proactive Relay Selection with Joint Impact of Hardware Impairment and Co-channel Interference

In this paper, we investigate the end-to-end performance of dual-hop proactive decode-and-forward relaying networks with Nth best relay selection in the presence of two practical deleterious effects: i) hardware impairment and ii) cochannel interference. In particular, we derive new exact and asymptotic closed-form expressions for the outage probability and average channel capacity of Nth best partial and opportunistic relay selection schemes over Rayleigh fading channels. Insightful discussions are provided. It is shown that, when the system cannot select the best relay for cooperation, the partial relay selection scheme outperforms the opportunistic method under the impact of the same co-channel interference (CCI). In addition, without CCI but under the effect of hardware impairment, it is shown that both selection strategies have the same asymptotic channel capacity. Monte Carlo simulations are presented to corroborate our analysis.

Unidirectional suppression of hydrogen oxidation on oxidized platinum clusters

Solar-driven water splitting to produce hydrogen may be an ideal solution for global energy and environment issues. Among the various photocatalytic systems, platinum has been widely used to co-catalyse the reduction of protons in water for hydrogen evolution. However, the undesirable hydrogen oxidation reaction can also be readily catalysed by metallic platinum, which limits the solar energy conversion efficiency in artificial photosynthesis. Here we report that the unidirectional suppression of hydrogen oxidation in photocatalytic water splitting can be fulfilled by controlling the valence state of platinum; this platinum-based cocatalyst in a higher oxidation state can act as an efficient hydrogen evolution site while suppressing the undesirable hydrogen back-oxidation. The findings in this work may pave the way for developing other high-efficientcy platinum-based catalysts for photocatalysis, photoelectrochemistry, fuel cells and water-gas shift reactions.

RNA interference in adult Ascaris suum - an opportunity for the development of a functional genomics platform that supports organism-, tissue- and cell-based biology in a nematode parasite

The sustainable control of animal parasitic nematodes requires the development of efficient functional genomics platforms to facilitate target validation and enhance anthelmintic discovery. Unfortunately, the utility of RNA interference (RNAi) for the validation of novel drug targets in nematode parasites remains problematic. Ascaris suum is an important veterinary parasite and a zoonotic pathogen. Here we show that adult A. suum is RNAi competent, and highlight the induction, spread and consistency of RNAi across multiple tissue types. This platform provides a new opportunity to undertake whole organism-, tissue- and cell-level gene function studies to enhance target validation processes for nematode parasites of veterinary/medical significance.

Suppression of the high affinity phosphate uptake system:A mechanism of arsenate tolerance in Holcus lanatus L.

In Holcus lanatus L. phosphate and arsenate are taken up by the same transport system. Short-term uptake kinetics of the high affinity arsenate transport system were determined in excised roots of arsenate-tolerant and non-tolerant genotypes. In tolerant plants the V_max of ion uptake in plants grown in phosphate-free media was decreased compared to non-tolerant plants, and the affinity of the uptake system was lower than in the non-tolerant plants. Both the reduction in V_max and the increase in K_m led to reduced arsenate influx into tolerant roots. When the two genotypes were grown in nutrient solution containing high levels of phosphate, there was little change in the uptake kinetics in tolerant plants. In non-tolerant plants, however, there was a marked decrease in the V_max to the level of the tolerant plants but with little change in the K_m. This suggests that the low rate of arsenate uptake over a wide range of differing root phosphate status is due to loss of induction of the synthesis of the arsenate (phosphate) carrier. © 1992 Oxford University Press.

Calmodulin disruption impacts growth and motility in juvenile liver fluke

Background: Deficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites. Calmodulins (CaMs) are small calcium-sensing proteins with ubiquitous expression in all eukaryotic organisms and generally use fluctuations in intracellular calcium levels to modulate cell signalling events. CaMs are essential for fundamental processes including the phosphorylation of protein kinases, gene transcription, calcium transport and smooth muscle contraction. In the blood fluke Schistosoma mansoni, calmodulins have been implicated in egg hatching, miracidial transformation and larval development. Previously, CaMs have been identified amongst liver fluke excretory-secretory products and three CaM-like proteins have been characterised biochemically from adult Fasciola hepatica, although their functions remain unknown.

Methods: In this study, we set out to investigate the biological function and control target potential of F. hepatica CaMs (FhCaMs) using RNAi methodology alongside novel in vitro bioassays.

Results: Our results reveal that: (i) FhCaMs are widely expressed in parenchymal cells throughout the forebody region of juvenile fluke; (ii) significant transcriptional knockdown of FhCaM1-3 was inducible by exposure to either long (~200 nt) double stranded (ds) RNAs or 27 nt short interfering (si) RNAs, although siRNAs were less effective than long dsRNAs; (iii) transient long dsRNA exposure-induced RNA interference (RNAi) of FhCaMs triggered transcript knockdown that persisted for ≥ 21 days, and led to detectable suppression of FhCaM proteins; (iv) FhCaM RNAi significantly reduced the growth of juvenile flukes maintained in vitro; (v) FhCaM RNAi juveniles also displayed hyperactivity encompassing significantly increased migration; (vi) both the reduced growth and increased motility phenotypes were recapitulated in juvenile fluke using the CaM inhibitor trifluoperazine hydrochloride, supporting phenotype specificity.

Conclusions: These data indicate that the Ca(2+)-modulating functions of FhCaMs are important for juvenile fluke growth and movement and provide the first functional genomics-based example of a growth-defect resulting from gene silencing in liver fluke. Whilst the phenotypic impacts of FhCaM silencing on fluke behaviour do not strongly support their candidature as new flukicide targets, the growth impacts encourage further consideration, especially in light of the speed of juvenile fluke growth in vivo.

Time-dependent electron interference prior to ionization in the hydrogen atom and hydrogen molecular ion

We investigate electron dynamics in the hydrogen atom and the hydrogen molecular ion when exposed to long wavelength laser pulses yet having intensity insufficient to ionize the system. We find that the field is still able to drive the electron, leading to time-dependent interference effects.

Exogenous RNA interference exposes contrasting roles for sugar exudation in host-finding by plant pathogens

Plant parasitic nematodes (PPN) locate host plants by following concentration gradients of root exudate chemicals in the soil. We present a simple method for RNA interference (RNAi)-induced knockdown of genes in tomato seedling roots, facilitating the study of root exudate composition, and PPN responses. Knockdown of sugar transporter genes, STP1 and STP2, in tomato seedlings triggered corresponding reductions of glucose and fructose, but not xylose, in collected root exudate. This corresponded directly with reduced infectivity and stylet thrusting of the promiscuous PPN Meloidogyne incognita, however we observed no impact on the infectivity or stylet thrusting of the selective Solanaceae PPN Globodera pallida. This approach can underpin future efforts to understand the early stages of plant-pathogen interactions in tomato and potentially other crop plants.