Alterations in predicted regulatory and coding regions of the sterol 14α-demethylase gene (CYP51) confer decreased azole sensitivity in the oilseed rape pathogen Pyrenopeziza brassicae

The incidence and severity of light leaf spot epidemics caused by the ascomycete fungus Pyrenopeziza brassicae on UK oilseed rape crops is increasing. The disease is currently controlled by a combination of host resistance, cultural practices and fungicide applications. We report decreases in sensitivities of modern UK P. brassicae isolates to the azole (imidazole and triazole) class of fungicides. By cloning and sequencing the P. brassicae CYP51 (PbCYP51) gene, encoding the azole target sterol 14α-demethylase, we identified two non-synonymous mutations encoding substitutions G460S and S508T associated with reduced azole sensitivity. We confirmed the impact of the encoded PbCYP51 changes on azole sensitivity and protein activity by heterologous expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a controllable promoter of native CYP51 expression. In addition, we identified insertions in the predicted regulatory regions of PbCYP51 in isolates with reduced azole sensitivity. The presence of these insertions was associated with enhanced transcription of PbCYP51 in response to sub-inhibitory concentrations of the azole fungicide tebuconazole. Genetic analysis of in vitro crosses of sensitive and resistant isolates confirmed the impact of PbCYP51 alterations in coding and regulatory sequences on a reduced sensitivity phenotype, as well as identifying a second major gene at another locus contributing to resistance in some isolates. The least sensitive field isolates carry combinations of upstream insertions and non-synonymous mutations, suggesting PbCYP51 evolution is on-going and the progressive decline in azole sensitivity of UK P. brassicae populations will continue. The implications for the future control of light leaf spot are discussed.

Paralog re-emergence: a novel, historically contingent mechanism in the evolution of antimicrobial resistance

Evolution of resistance to drugs and pesticides poses a serious threat to human health and agricultural production. CYP51 encodes the target site of azole fungicides, widely used clinically and in agriculture. Azole resistance can evolve due to point mutations or overexpression of CYP51, and previous studies have shown that fungicide-resistant alleles have arisen by de novo mutation. Paralogs CYP51A and CYP51B are found in filamentous ascomycetes, but CYP51A has been lost from multiple lineages. Here, we show that in the barley pathogen Rhynchosporium commune, re-emergence of CYP51A constitutes a novel mechanism for the evolution of resistance to azoles. Pyrosequencing analysis of historical barley leaf samples from a unique long-term experiment from 1892 to 2008 indicates that the majority of the R. commune population lacked CYP51A until 1985, after which the frequency of CYP51A rapidly increased. Functional analysis demonstrates that CYP51A retains the same substrate as CYP51B, but with different transcriptional regulation. Phylogenetic analyses show that the origin of CYP51A far predates azole use, and newly sequenced Rhynchosporium genomes show CYP51A persisting in the R. commune lineage rather than being regained by horizontal gene transfer; therefore, CYP51A re-emergence provides an example of adaptation to novel compounds by selection from standing genetic variation.

Poetry on Pre-Raphaelite principles: science, nature and knowledge in William Michael Rossetti’s 'Fancies at Leisure' and 'Mrs. Holmes Grey'

This article demonstrates how early Pre-Raphaelite poetry worked according to the principle that art should be modelled on science theorised by the Pre-Raphaelites in their early essays. As the main theorists (rather than practitioners) of Pre-Raphaelite art, F. G. Stephens and William Michael Rossetti defined the Pre-Raphaelite project in terms of observation, investigation, experiment, the “adherence to fact” and the “search after truth”. In the hands of the early Pre-Raphaelite poets, and particularly Rossetti himself, poetry too becomes a mode of scientific enquiry into the natural world, the nature of observation, human psychology and medical practice.

Study of picosecond processes of an intercalated dipyridophenazine Cr(iii) complex bound to defined sequence DNAs using transient absorption and time-resolved infrared methods

Picosecond transient absorption (TA) and time-resolved infrared (TRIR) measurements of rac-[Cr(phen)2(dppz)]3+ (1) intercalated into double-stranded guanine-containing DNA reveal that the excited state is very rapidly quenched. As no evidence was found for the transient electron transfer products, it is proposed that the back electron transfer reaction must be even faster (<3 ps).

Enantiomeric conformation controls rate and yield of photoinduced electron transfer in DNA sensitized by Ru(II) Dipyridophenazine complexes

Photosensitized oxidation of guanine is an important route to DNA damage. Ruthenium polypyridyls are very useful photosensitizers as their reactivity and DNA-binding properties are readily tunable. Here we show a strong difference in the reactivity of the two enantiomers of [Ru(TAP)2(dppz)]2+, by using time-resolved visible and IR spectroscopy. This reveals that the photosensitized one-electron oxidation of guanine in three oligonucleotide sequences proceeds with similar rates and yields for bound delta-[Ru(TAP)2(dppz)]2+, whereas those for the lambda enantiomer are very sensitive to base sequence. It is proposed that these differences are due to preferences of each enantiomer for different binding sites in the duplex.

Reversal of a single base pair step controls guanine photo-oxidation by an intercalating Ru(II) dipyridophenazine complex

Small changes in DNA sequence can often have major biological effects. Here the rates and yields of guanine photo-oxidation by Λ [Ru(TAP)2(dppz)]2+ have been compared in 5′-{CCGGATCCGG}2 and 5′-{CCGGTACCGG}2 using ps/ns transient visible and time-resolved IR (TRIR) spectroscopy. The inefficiency of electron transfer in the TA sequence is consistent with the 5′-TA-3′ vs. 5′-AT-3′ binding preference predicted by X-ray crystallography. The TRIR spectra also reveal the differences in binding sites in the two oligonucleotides.

Monitoring guanine photo-oxidation by enantiomerically resolved Ru(II) dipyridophenazine complexes using inosine-substituted oligonucleotides

The intercalating [Ru(TAP)2(dppz)]2+ complex can photo-oxidise guanine in DNA, although in mixed-sequence DNA it can be difficult to understand the precise mechanism due to uncertainties in where and how the complex is bound. Replacement of guanine with the less oxidisable inosine (I) base can be used to understand the mechanism of electron transfer (ET). Here the ET has been compared for both L- and D-enantiomers of [Ru(TAP)2(dppz)]2+ in a set of sequences where guanines in the readily oxidisable GG step in {TCGGCGCCGA}2 have been replaced with I. The ET has been monitored using picosecond and nanosecond transient absorption and ps-time-resolved IR spectroscopy. In both cases inosine replacement leads to a diminished yield, but the trends are strikingly different for L- and D-complexes.

Looking back, moving forward: drama, disability and social justice

The aim of this chapter is to briefly outline how disability has been represented in theatre, what access disabled people have had to drama and theatre in the past, and what might be achieved in the pursuit of social justice with young people in relation to awareness of and provision for disability. It will focus in particular on how disability has been addressed in drama education and what assumptions have been made regarding drama and disability in education. In considering such issues one might perceive manifestations of what Freebody and Finneran (2013) recognise as an overlapping and ‘somewhat artificially created dichotomy between drama for social justice and drama about social justice.’ This chapter will examine some examples of how drama has been used to give students in mainstream schools insights into disability, and the philosophy that underpins the drama curriculum of one special school where the focus is on drama as social justice: the argument being that in some cases simply doing drama is, in effect, a manifestation of social justice. Finally, some of the progress made in recent years regarding access and engagement will be addressed through specific reference to the authors’ on-going work into ‘performing social research’ (Shah, 2013) and how theatres are increasingly attempting to give more access to disabled young people and their families by offering ‘relaxed performances.’

Monitoring one-electron photo-oxidation of guanine in DNA crystals using ultrafast infrared spectroscopy

To understand the molecular origins of diseases caused by ultraviolet and visible light, and also to develop photodynamic therapy, it is important to resolve the mechanism of photoinduced DNA damage. Damage to DNA bound to a photosensitizer molecule frequently proceeds by one-electron photo-oxidation of guanine, but the precise dynamics of this process are sensitive to the location and the orientation of the photosensitizer, which are very difficult to define in solution. To overcome this, ultrafast time-resolved infrared (TRIR) spectroscopy was performed on photoexcited ruthenium polypyridyl–DNA crystals, the atomic structure of which was determined by X-ray crystallography. By combining the X-ray and TRIR data we are able to define both the geometry of the reaction site and the rates of individual steps in a reversible photoinduced electron-transfer process. This allows us to propose an individual guanine as the reaction site and, intriguingly, reveals that the dynamics in the crystal state are quite similar to those observed in the solvent medium.

Rotational aesthetics: Michael Bay and contemporary cinema’s machine movement

Digital imaging technologies enable a mastery of the visual that in recent mainstream cinema frequently manifests as certain kinds of spatial reach, orientation and motion. In such a context Michael Bay’s Transformers franchise can be framed as a digital re-tooling of a familiar fantasy of vehicular propulsion, US car culture writ large in digitally crafted spectacles of diegetic speed, the vehicular chase film ‘2.0’. Movement is central to these films, calling up Scott Bukatman’s observation that in spectacular visual media ‘movement has become more than a tool of bodily knowledge; it has become an end in itself’ (2003: 125). Not all movements and not all instances of vehicular propulsion are the same however. How might we evaluate what is at stake in a film’s assertion of movement as an end in itself, and the form that assertion takes, its articulations of diegetic velocity, corporeality, and spatial penetration? Deploying an attentiveness towards the specificity of aesthetic detail and affective impact in Bay’s delineation of movement, this essay suggests that the franchise poses questions about the relationship of human movement to machine movement that exceed their narrative basis. Identifying a persistent rotational trope in the franchise that in its audio-visual articulation combines oddly anachronistic elements (evoking the mechanical rather than the digital), the article argues that the films prioritise certain fantasies of transformation and spatial penetration, and certain modes of corporeality, as one response to contemporary debates about digital technologisation, sustainable energy, and cinematic spectacle. In this way the franchise also represents a particular moment in a more widely discernible preoccupation in contemporary cinema with what we might call a ‘rotational aesthetics’ of action, a machine movement made possible by the digital, but which invokes earlier histories and fantasies of animation, propulsion, mechanization and mechanization to particular ends.

The COnvective Precipitation Experiment (COPE): investigating the origins of heavy precipitation in the southwestern UK

A recent field campaign in southwest England used numerical modeling integrated with aircraft and radar observations to investigate the dynamic and microphysical interactions that can result in heavy convective precipitation. The COnvective Precipitation Experiment (COPE) was a joint UK-US field campaign held during the summer of 2013 in the southwest peninsula of England, designed to study convective clouds that produce heavy rain leading to flash floods. The clouds form along convergence lines that develop regularly due to the topography. Major flash floods have occurred in the past, most famously at Boscastle in 2004. It has been suggested that much of the rain was produced by warm rain processes, similar to some flash floods that have occurred in the US. The overarching goal of COPE is to improve quantitative convective precipitation forecasting by understanding the interactions of the cloud microphysics and dynamics and thereby to improve NWP model skill for forecasts of flash floods. Two research aircraft, the University of Wyoming King Air and the UK BAe 146, obtained detailed in situ and remote sensing measurements in, around, and below storms on several days. A new fast-scanning X-band dual-polarization Doppler radar made 360-deg volume scans over 10 elevation angles approximately every 5 minutes, and was augmented by two UK Met Office C-band radars and the Chilbolton S-band radar. Detailed aerosol measurements were made on the aircraft and on the ground. This paper: (i) provides an overview of the COPE field campaign and the resulting dataset; (ii) presents examples of heavy convective rainfall in clouds containing ice and also in relatively shallow clouds through the warm rain process alone; and (iii) explains how COPE data will be used to improve high-resolution NWP models for operational use.