Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry

RNA interference has provided valuable insight into a wide range of biological systems and is a powerful tool for the analysis of gene function. The exploitation of this pathway to block the expression of specific gene targets holds considerable promise for the development of novel RNAi-based insect management strategies. In addition, there are a wide number of future potential applications of RNAi to control agricultural insect pests as well as its use for prevention of diseases in beneficial insects. The potential to synthesise large quantities of dsRNA by in-vitro transcription or in bacterial systems for RNA interference applications has generated significant demand for the development and application of high throughput analytical tools for the rapid extraction, purification and analysis of dsRNA. Here we have developed analytical methods that enable the rapid purification of dsRNA from associated impurities from bacterial cells in conjunction with downstream analyses. We have optimised TRIzol extractions in conjunction with a single step protocol to remove contaminating DNA and ssRNA, using RNase T1/DNase I digestion under high-salt conditions in combination with solid phase extraction to purify the dsRNA. In addition, we have utilised and developed IP RP HPLC for the rapid, high resolution analysis of the dsRNA. Furthermore, we have optimised base-specific cleavage of dsRNA by RNase A and developed a novel method utilising RNase T1 for RNase mass mapping approaches to further characterise the dsRNA using liquid chromatography interfaced with mass spectrometry

Determining the Solution Space of Vertex-Cover by Interactions and Backbones

To solve the combinatorial optimization problems especially the minimal Vertex-cover problem with high efficiency, is a significant task in theoretical computer science and many other subjects. Aiming at detecting the solution space of Vertex-cover, a new structure named interaction between nodes is defined and discovered for random graph, which results in the emergence of the frustration and long-range correlation phenomenon. Based on the backbones and interactions with a node adding process, we propose an Interaction and Backbone Evolution Algorithm to achieve the reduced solution graph, which has a direct correspondence to the solution space of Vertex-cover. By this algorithm, the whole solution space can be obtained strictly when there is no leaf-removal core on the graph and the odd cycles of unfrozen nodes bring great obstacles to its efficiency. Besides, this algorithm possesses favorable exactness and has good performance on random instances even with high average degrees. The interaction with the algorithm provides a new viewpoint to solve Vertex-cover, which will have a wide range of applications to different types of graphs, better usage of which can lower the computational complexity for solving Vertex-cover

The determinants of election to the United Nations Security Council

This is the author's accepted manuscript. The final publication is available at Springer via http://dx.doi.org/10.1007/s11127-013-0096-4.The United Nations Security Council (UNSC) is the foremost international body responsible for the maintenance of international peace and security. Members vote on issues of global importance and consequently receive perks—election to the UNSC predicts, for instance, World Bank and IMF loans. But who gets elected to the UNSC? Addressing this question empirically is not straightforward as it requires a model that allows for discrete choices at the regional and international levels; the former nominates candidates while the latter ratifies them. Using an original multiple discrete choice model to analyze a dataset of 180 elections from 1970 to 2005, we find that UNSC election appears to derive from a compromise between the demands of populous countries to win election more frequently and a norm of giving each country its turn. We also find evidence that richer countries from the developing world win election more often, while involvement in warfare lowers election probability. By contrast, development aid does not predict election

Optimizing the production of dsRNA biocontrols in microbial systems using multiple transcriptional terminators

Crop pests and pathogens annually cause over $220 billion in global crop damage, with insects consuming 5%–20% of major grain crops. Current crop pest and disease control strategies rely on insecticidal and fungicidal sprays, plant genetic resistance, transgenes, and agricultural practices. Double-stranded RNA (dsRNA) is emerging as a novel sustainable method of plant protection as an alternative to traditional chemical pesticides. Successful commercialization of dsRNA-based biocontrols requires the economical production of large quantities of dsRNA combined with suitable delivery methods to ensure RNAi efficacy against the target pest. In this study, we have optimized the design of plasmid DNA constructs to produce dsRNA biocontrols in Escherichia coli, by employing a wide range of alternative synthetic transcriptional terminators before measurement of dsRNA yield. We demonstrate that a 7.8-fold increase of dsRNA was achieved using triple synthetic transcriptional terminators within a dual T7 dsRNA production system compared to the absence of transcriptional terminators. Moreover, our data demonstrate that batch fermentation production dsRNA using multiple transcriptional terminators is scalable and generates significantly higher yields of dsRNA generated in the absence of transcriptional terminators at both small-scale batch culture and large-scale fermentation. In addition, we show that application of these dsRNA biocontrols expressed in E. coli cells results in increased insect mortality. Finally, novel mass spectrometry analysis was performed to determine the precise sites of transcriptional termination at the different transcriptional terminators providing important further mechanistic insight

Molecular Determinants of S100B Oligomer Formation

Background: S100B is a dimeric protein that can form tetramers, hexamers and higher order oligomers. These forms have been suggested to play a role in RAGE activation. Methodology/Principal Findings: Oligomerization was found to require a low molecular weight trigger/cofactor and could not be detected for highly pure dimer, irrespective of handling. Imidazol was identified as a substance that can serve this role. Oligomerization is dependent on both the imidazol concentration and pH, with optima around 90 mM imidazol and pH 7, respectively. No oligomerization was observed above pH 8, thus the protonated form of imidazol is the active species in promoting assembly of dimers to higher species. However, disulfide bonds are not involved and the process is independent of redox potential. The process was also found to be independent of whether Ca 2+ is bound to the protein or not. Tetramers that are purified from dimers and imidazol by gel filtration are kinetically stable, but dissociate into dimers upon heating. Dimers do not revert to tetramer and higher oligomer unless imidazol is again added. Both tetramers and hexamers bind the target peptide from p53 with retained stoichiometry of one peptide per S100B monomer, and with high affinity (lgK = 7.360.2 and 7.260.2, respectively in 10 mM BisTris, 5 mM CaCl 2, pH 7.0), which is less than one order of magnitude reduced compared to dimer under the same buffer conditions. Conclusion/Significance: S100B oligomerization requires protonated imidazol as a trigger/cofactor. Oligomers ar

Evaluation of the Arabin cervical pessary for prevention of preterm birth in women with a twin pregnancy and short cervix (STOPPIT-2):An open-label randomised trial and updated meta-analysis

BackgroundPreterm-labour-associated preterm birth is a common cause of perinatal mortality and morbidity in twin pregnancy. We aimed to test the hypothesis that the Arabin pessary would reduce preterm-labour-associated preterm birth by 40% or greater in women with a twin pregnancy and a short cervix.Methods and findingsWe conducted an open-label randomised controlled trial in 57 hospital antenatal clinics in the UK and Europe. From 1 April 2015 to 14 February 2019, 2,228 women with a twin pregnancy underwent cervical length screening between 18 weeks 0 days and 20 weeks 6 days of gestation. In total, 503 women with cervical length ≤ 35 mm were randomly assigned to pessary in addition to standard care (n = 250, mean age 32.4 years, mean cervical length 29 mm, with pessary inserted in 230 women [92.0%]) or standard care alone (n = 253, mean age 32.7 years, mean cervical length 30 mm). The pessary was inserted before 21 completed weeks of gestation and removed at between 35 and 36 weeks or before birth if earlier. The primary obstetric outcome, spontaneous onset of labour and birth before 34 weeks 0 days of gestation, was present in 46/250 (18.4%) in the pessary group compared to 52/253 (20.6%) following standard care alone (adjusted odds ratio [aOR] 0.87 [95% CI 0.55-1.38], p = 0.54). The primary neonatal outcome-a composite of any of stillbirth, neonatal death, periventricular leukomalacia, early respiratory morbidity, intraventricular haemorrhage, necrotising enterocolitis, or proven sepsis, from birth to 28 days after the expected date of delivery-was present in 67/500 infants (13.4%) in the pessary group compared to 76/506 (15.0%) following standard care alone (aOR 0.86 [95% CI 0.54-1.36], p = 0.50). The positive and negative likelihood ratios of a short cervix (≤35 mm) to predict preterm birth before 34 weeks were 2.14 and 0.83, respectively. A meta-analysis of data from existing publications (4 studies, 313 women) and from STOPPIT-2 indicated that a cervical pessary does not reduce preterm birth before 34 weeks in women with a short cervix (risk ratio 0.74 [95% CI 0.50-1.11], p = 0.15). No women died in either arm of the study; 4.4% of babies in the Arabin pessary group and 5.5% of babies in the standard treatment group died in utero or in the neonatal period (p = 0.53). Study limitations include lack of power to exclude a smaller than 40% reduction in preterm labour associated preterm birth, and to be conclusive about subgroup analyses.ConclusionsThese results led us to reject our hypothesis that the Arabin pessary would reduce the risk of the primary outcome by 40%. Smaller treatment effects cannot be ruled out.Trial registrationISRCTN Registry ISRCTN 02235181. ClinicalTrials.gov NCT02235181