Synthesis of 3 '-S-phosphorothiolate oligonucleotides for their potential use in RNA interference

The potency of RNA interference (RNAi) undoubtedly can be improved through chemical modifications to the small interfering RNAs (siRNA). By incorporation of the 3′-S-phosphorothiolate modification into strands of RNA, it is hoped that specific regions of a siRNA duplex can be stabilised to enhance the target binding affinity of a selected antisense strand into the activated RNA-induced silencing complex (RISC*). Oligonucleotides composed entirely of this modification are desirable so unconventional 5′ → 3′ synthesis is investigated, with initial solution-phase testing proving successful. The phosphoroamidite monomer required for solid-phase synthesis has also been produced.

RNA interference suppression of genes in glycosyl transferase families 43 and 47 in wheat starchy endosperm causes large decreases in arabinoxylan content

The cell walls of wheat (Triticum aestivum) starchy endosperm are dominated by arabinoxylan (AX), accounting for 65% to 70% of the polysaccharide content. Genes within two glycosyl transferase (GT) families, GT43 (IRREGULAR XYLEM9 [IRX9] and IRX14) and GT47 (IRX10), have previously been shown to be involved in the synthesis of the xylan backbone in Arabidopsis, and close homologs of these have been implicated in the synthesis of xylan in other species. Here, homologs of IRX10 TaGT47_2 and IRX9 TaGT43_2, which are highly expressed in wheat starchy endosperm cells, were suppressed by RNA interference (RNAi) constructs driven by a starchy endosperm-specific promoter. The total amount of AX was decreased by 40% to 50% and the degree of arabinosylation was increased by 25% to 30% in transgenic lines carrying either of the transgenes. The cell walls of starchy endosperm in sections of grain from TaGT43_2 and TaGT47_2 RNAi transgenics showed decreased immunolabeling for xylan and arabinoxylan epitopes and approximately 50% decreased cell wall thickness compared with controls. The proportion of AX that was water soluble was not significantly affected, but average AX polymer chain length was decreased in both TaGT43_2 and TaGT47_2 RNAi transgenics. However, the long AX chains seen in controls were absent in TaGT43_2 RNAi transgenics but still present in TaGT47_2 RNAi transgenics. The results support an emerging picture of IRX9-like and IRX10-like proteins acting as key components in the xylan synthesis machinery in both dicots and grasses. Since AX is the main component of dietary fiber in wheat foods, the TaGT43_2 and TaGT47_2 genes are of major importance to human nutrition.

Antisense-induced myostatin exon skipping leads to muscle hypertrophy in mice following Octa‑guanidine morpholino oligomer treatment

Myostatin is a negative regulator of muscle mass, and several strategies are being developed to knockdown its expression to improve muscle-wasting conditions. Strategies using antimyostatin-blocking antibodies, inhibitory-binding partners, signal transduction blockers, and RNA interference system (RNAi)-based knockdown have yielded promising results and increased muscle mass in experimental animals. These approaches have, however, a number of disadvantages such as transient effects or adverse immune complications. We report here the use of antisense oligonucleotides (AOs) to manipulate myostatin pre-mRNA splicing and knockdown myostatin expression. Both 2’O-methyl phosphorothioate RNA (2’OMePS) and phosphorodiamidate morpholino oligomers (PMO) led to efficient exon skipping in vitro and in vivo and knockdown of myostatin at the transcript level. The substantial myostatin exon skipping observed after systemic injection of Vivo-PMO into normal mice led to a significant increase in soleus muscle mass as compared to the controls injected with normal saline suggesting that this approach could be feasible to ameliorate muscle-wasting pathologies.

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

The genome strikes back: The evolutionary importance of defence against mobile elements

Increasingly, we regard the genome as a site and source of genetic conflict. This fascinating 'bottom-up' view brings up appealing connections between genome biology and whole-organism ecology, in which populations of elements compete with one another in their genomic habitat. Unlike other habitats, though, a host genome has its own evolutionary interests and is often able to defend itself against molecular parasites. Most well-studied organisms employ strategies to protect their genomes against the harmful effects of genomic parasites, including methylation, various pathways of RNA interference, and more unusual tricks such as repeat induced point-mutation (RIP). These genome defence systems are not obscure biological curiosities, but fundamentally important to the integrity and cohesion of the genome, and exert a powerful influence on genome evolution.

Plant U13 orthologues and orphan snoRNAs identified by RNomics of RNA from Arabidopsis nucleoli

Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and uncapped small RNAs using RNA from isolated nucleoli from Arabidopsis cell cultures. We have identified 31 novel snoRNA genes (9 box C/D and 22 box H/ACA) and 15 new variants of previously described snoRNAs. Three related capped snoRNAs with a distinct gene organization and structure were identified as orthologues of animal U13snoRNAs. In addition, eight of the novel genes had no complementarity to rRNAs or snRNAs and are therefore putative orphan snoRNAs potentially reflecting wider functions for these RNAs. The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization. The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters. These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants.

Interference and communications among active network applications

This paper focuses on active networks applications and in particular on the possible interactions among these applications. Active networking is a very promising research field which has been developed recently, and which poses several interesting challenges to network designers. A number of proposals for e±cient active network architectures are already to be found in the literature. However, how two or more active network applications may interact has not being investigated so far. In this work, we consider a number of applications that have been designed to exploit the main features of active networks and we discuss what are the main benefits that these applications may derive from them. Then, we introduce some forms of interaction including interference and communications among applications, and identify the components of an active network architecture that are needed to support these forms of interaction. We conclude by presenting a brief example of an active network application exploiting the concept of interaction.

Interference competition and high temperature reduce the ‘virulence’ of fig wasps and contribute to stability of a fig-wasp mutualism

Fig trees are pollinated by fig wasps, which also oviposit in female flowers. The wasp larvae gall and eat developing seeds. Although fig trees benefit from allowing wasps to oviposit, because the wasp offspring disperse pollen, figs must prevent wasps from ovipositing in all flowers, or seed production would cease, and the mutualism would go extinct. In Ficus racemosa, we find that syconia (‘figs’) that have few foundresses (ovipositing wasps) are underexploited in the summer (few seeds, few galls, many empty ovules) and are overexploited in the winter (few seeds, many galls, few empty ovules). Conversely, syconia with many foundresses produce intermediate numbers of galls and seeds, regardless of season. We use experiments to explain these patterns, and thus, to explain how this mutualism is maintained. In the hot summer, wasps suffer short lifespans and therefore fail to oviposit in many flowers. In contrast, cooler temperatures in the winter permit longer wasp lifespans, which in turn allows most flowers to be exploited by the wasps. However, even in winter, only in syconia that happen to have few foundresses are most flowers turned into galls. In syconia with higher numbers of foundresses, interference competition reduces foundress lifespans, which reduces the proportion of flowers that are galled. We further show that syconia encourage the entry of multiple foundresses by delaying ostiole closure. Taken together, these factors allow fig trees to reduce galling in the wasp-benign winter and boost galling (and pollination) in the wasp-stressing summer. Interference competition has been shown to reduce virulence in pathogenic bacteria. Our results show that interference also maintains cooperation in a classic, cooperative symbiosis, thus linking theories of virulence and mutualism. More generally, our results reveal how frequency-dependent population regulation can occur in the fig-wasp mutualism, and how a host species can ‘set the rules of the game’ to ensure mutualistic behavior in its symbionts.

A simple and rapid method for preserving RNA of aquatic invertebrates for ecotoxicogenomics

Here we describe a novel, inexpensive and simple method for preserving RNA that reduces handling stress in aquatic invertebrates following ecotoxicogenomic experimentation. The application of the method is based on transcriptomic experiments conducted on Daphnia magna, but may easily be applied on a range of other aquatic organisms of a particular size with e.g. amphipod Gammarus pulex representing an upper size limit. We explain in detail how to apply this new method, named the "Cylindrical Sieve (CS) system", and highlight its advantages and disadvantages.

Mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in RNA binding by using surface plasmon resonance

Phosphorylation of the coronavirus nucleoprotein (N protein) has been predicted to play a role in RNA binding. To investigate this hypothesis, we examined the kinetics of RNA binding between nonphosphorylated and phosphorylated infectious bronchitis virus N protein with nonviral and viral RNA by surface plasmon resonance (Biacore). Mass spectroscopic analysis of N protein identified phosphorylation sites that were proximal to RNA binding domains. Kinetic analysis, by surface plasmon resonance, indicated that nonphospborylated N protein bound with the same affinity to viral RNA as phosphorylated N protein. However, phosphorylated N protein bound to viral RNA with a higher binding affinity than nonviral RNA, suggesting that phosphorylation of N protein determined the recognition of virus RNA. The data also indicated that a known N protein binding site (involved in transcriptional regulation) consisting of a conserved core sequence present near the 5' end of the genome (in the leader sequence) functioned by promoting high association rates of N protein binding. Further analysis of the leader sequence indicated that the core element was not the only binding site for N protein and that other regions functioned to promote high-affinity binding.

Factors required for the uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3D(pol)) in vitro

The 5' terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 313 (VTg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3D(pol). To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3D(pol) in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV ere has been identified previously to be within the 5' untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 313 peptides has now been determined, and the role of the FMDV ere (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.

An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro

G-protein-coupled receptors are desensitized by a two-step process. In a first step, G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-activated receptors that subsequently bind to a second class of proteins, the arrestins. GRKs can be classified into three subfamilies, which have been implicated in various diseases. The physiological role(s) of GRKs have been difficult to study as selective inhibitors are not available. We have used SELEX (systematic evolution of ligands by exponential enrichment) to develop RNA aptamers that potently and selectively inhibit GRK2. This process has yielded an aptamer, C13, which bound to GRK2 with a high affinity and inhibited GRK2-catalyzed rhodopsin phosphorylation with an IC50 of 4.1 nM. Phosphorylation of rhodopsin catalyzed by GRK5 was also inhibited, albeit with 20-fold lower potency (IC50 of 79 nM). Furthermore, C13 reveals significant specificity, since almost no inhibitory activity was detectable testing it against a panel of 14 other kinases. The aptamer is two orders of magnitude more potent than the best GRK2 inhibitors described previously and shows high selectivity for the GRK family of protein kinases.

Changing patterns of cognitive-motor interference (CMI) over time during recovery from stroke

Objective: To explore the extent and nature of change in cognitive-motor interference (CMI) among rehabilitating stroke patients who showed dual-task gait decrement at initial assessment. Design: Experimental, with in-subjects, repeated measures design. Setting: Rehabilitation centre for adults with acquired, nonprogressive brain injury. Subjects: Ten patients with unilateral stroke, available for reassessment 1-9 months following their participation in a study of CMI after brain injury. Measures: Median stride duration; mean word generation. Methods: Two x one-minute walking trials, two x one-minute word generation trials, two x one-minute trials of simultaneous walking and word generation; 10-metre walking time; Barthel ADL Scale score. Results: Seven out of ten patients showed reduction over time in dual-task gait decrement. Three out of ten showed reduction in cognitive decrement. Only one showed concomitant reduction in gait and word generation decrement. Conclusion: Extent of CMI during relearning to walk after a stroke reduced over time in the majority of patients. Effects were more evident in improved stride duration than improved cognitive performance. Measures of multiple task performance should be included in assessment for functional recovery.

Interference filters and coatings for mid-infrared astronomy (8-30µm)

The VISIR instrument for the European Southern Observatory (ESO) Very Large Telescope (VLT) is a thermal-infrared imager and spectrometer currently being developed by the French Service d'Astrophysique of CEA Saclay, and Dutch NFRA ASTRON Dwingeloo consortium. This cryogenic instrument will employ precision infrared bandpass filters in the N-( =7.5-14µm) and Q-( =16-28µm) band mid-IR atmospheric windows to study interstellar and circumstellar environments crucial for star and planetary formation theories. As the filters in these mid-IR wavelength ranges are of interest to many astronomical cryogenic instruments, a worldwide astronomical filter consortium was set up with participation from 12 differing institutes, each requiring instrument specific filter operating environments and optical metrology. This paper describes the design and fabrication methods used to manufacture these astronomical consortium filters, including the rationale for the selection of multilayer coating designs, temperature-dependant optical properties of the filter materials and FTIR spectral measurements showing the changes in passband and blocking performance on cooling to <50K. We also describe the development of a 7-14µm broadband antireflection coating deposited on Ge lenses and KRS-5 grisms for cryogenic operation at 40K