Reflections on Beckett: a centenary celebration

The essays in this collection were originally delivered as part of the Samuel Beckett lecture series at Trinity College Dublin, Samuel Beckett's alma mater. The contributors include eminent Beckett scholars such as Linda Ben-Zvi, Enoch Brater, Ruby Cohn and Stan Gontarski, theatre scholars such as Herbert Blau and Joseph Roach, practitioners such as the Irish actor, Barry McGovern, and cultural critics such as Marina Warner and Terry Eagleton. The collection sheds new light on Beckett's enigmatic theater, offering new perspectives on Beckett's use of language and silence, on his attitudes toward the body, on those who influenced him and on those he has influenced (including Suzan-Lori Parks and Femi Osofisan), and on Beckett and the art of self-collaboration. Each contributor places the playwright into a network of genealogies and legacies and his work into important historical, cultural, and aesthetic contexts. Together, the essays demonstrate Beckett's impact on theater, performance, and visual arts during the latter half of the twentieth century and serve to open up new directions for Beckett studies.

Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity

Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine.

Detecting insect pollinator declines on regional and global scales.

Recently there has been considerable concern about declines in bee communities in agricultural and natural habitats. The value of pollination to agriculture, provided primarily by bees, is >$200 billion/year worldwide, and in natural ecosystems it is thought to be even greater. However, no monitoring program exists to accurately detect declines in abundance of insect pollinators; thus, it is difficult to quantify the status of bee communities or estimate the extent of declines. We used data from 11 multiyear studies of bee communities to devise a program to monitor pollinators at regional, national, or international scales. In these studies, 7 different methods for sampling bees were used and bees were sampled on 3 different continents. We estimated that a monitoring program with 200–250 sampling locations each sampled twice over 5 years would provide sufficient power to detect small (2–5%) annual declines in the number of species and in total abundance and would cost U.S.$2,000,000. To detect declines as small as 1% annually over the same period would require >300 sampling locations. Given the role of pollinators in food security and ecosystem function, we recommend establishment of integrated regional and international monitoring programs to detect changes in pollinator communities.

Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.

Bacillus subtilis natto: a non-toxic source of poly-γ-glutamic acid that could be used as a cryoprotectant for probiotic bacteria

It is common practice to freeze dry probiotic bacteria to improve their shelf life. However, the freeze drying process itself can be detrimental to their viability. The viability of probiotics could be maintained if they are administered within a microbially produced biodegradable polymer - poly-γ-glutamic acid (γ-PGA) - matrix. Although the antifreeze activity of γ-PGA is well known, it has not been used for maintaining the viability of probiotic bacteria during freeze drying. The aim of this study was to test the effect of γ-PGA (produced by B. subtilis natto ATCC 15245) on the viability of probiotic bacteria during freeze drying and to test the toxigenic potential of B. subtilis natto. 10% γ-PGA was found to protect Lactobacillus paracasei significantly better than 10% sucrose, whereas it showed comparable cryoprotectant activity to sucrose when it was used to protect Bifidobacterium breve and Bifidobacterium longum. Although γ-PGA is known to be non-toxic, it is crucial to ascertain the toxigenic potential of its source, B. subtilis natto. Presence of six genes that are known to encode for toxins were investigated: three component hemolysin (hbl D/A), three component non-haemolytic enterotoxin (nheB), B. cereus enterotoxin T (bceT), enterotoxin FM (entFM), sphingomyelinase (sph) and phosphatidylcholine-specific phospholipase (piplc). From our investigations, none of these six genes were present in B. subtilis natto. Moreover, haemolytic and lecithinase activities were found to be absent. Our work contributes a biodegradable polymer from a non-toxic source for the cryoprotection of probiotic bacteria, thus improving their survival during the manufacturing process.

Physiological, biochemical and transcriptional analysis of onion bulbs during storage

Abstract: During the transition from endo-dormancy to eco-dormancy and subsequent growth, the onion bulb undergoes the transition from sink organ to source, to sustain cell division in the meristematic tissue. The mechanisms controlling these processes are not fully understood. Here, a detailed analysis of whole onion bulb physiological, biochemical and transcriptional changes in response to sprouting is reported, enabling a better knowledge of the mechanisms regulating post-harvest onion sprout development. Biochemical and physiological analyses were conducted on different cultivars ('Wellington', 'Sherpa' and 'Red Baron') grown at different sites over 3 years, cured at different temperatures (20, 24 and 28 degrees C) and stored under different regimes (1, 3, 6 and 6 1 degrees C). In addition, the first onion oligonucleotide microarray was developed to determine differential gene expression in onion during curing and storage, so that transcriptional changes could support biochemical and physiological analyses. There were greater transcriptional differences between samples at harvest and before sprouting than between the samples taken before and after sprouting, with some significant changes occurring during the relatively short curing period. These changes are likely to represent the transition from endo-dormancy to sprout suppression, and suggest that endo-dormancy is a relatively short period ending just after curing. Principal component analysis of biochemical and physiological data identified the ratio of monosaccharides (fructose and glucose) to disaccharide (sucrose), along with the concentration of zeatin riboside, as important factors in discriminating between sprouting and pre-sprouting bulbs. These detailed analyses provide novel insights into key regulatory triggers for sprout dormancy release in onion bulbs and provide the potential for the development of biochemical or transcriptional markers for sprout initiation. Evidence presented herein also suggests there is no detrimental effect on bulb storage life and quality caused by curing at 20 degrees C, producing a considerable saving in energy and costs.

Ethylene and 1-methylcyclopropene differentially regulate gene expression during onion sprout suppression

Onion (Allium cepa) is regarded as a nonclimacteric vegetable. In onions, however, ethylene can suppress sprouting while the ethylene-binding inhibitor 1-methylcyclopropene (1-MCP) can also suppress sprout growth; yet, it is unknown how ethylene and 1-MCP elicit the same response. In this study, onions were treated with 10 mu L L(-1) ethylene or 1 mu L L(-1) 1-MCP individually or in combination for 24 h at 20 degrees C before or after curing (6 weeks) at 20 degrees C or 28 degrees C and then stored at 1 degrees C. Following curing, a subset of these same onions was stored separately under continuous air or ethylene (10 mu L L(-1)) at 1 degrees C. Onions treated with ethylene and 1-MCP in combination after curing for 24 h had reduced sprout growth as compared with the control 25 weeks after harvest. Sprout growth following storage beyond 25 weeks was only reduced through continuous ethylene treatment. This observation was supported by a higher proportion of down-regulated genes characterized as being involved in photosynthesis, measured using a newly developed onion microarray. Physiological and biochemical data suggested that ethylene was being perceived in the presence of 1-MCP, since sprout growth was reduced in onions treated with 1-MCP and ethylene applied in combination but not when applied individually. A cluster of probes representing transcripts up-regulated by 1-MCP alone but down-regulated by ethylene alone or in the presence of 1-MCP support this suggestion. Ethylene and 1-MCP both down-regulated a probe tentatively annotated as an ethylene receptor as well as ethylene-insensitive 3, suggesting that both treatments down-regulate the perception and signaling events of ethylene.

Response to zinc deficiency of two rice lines with contrasting tolerance is determined by root growth maintenance and organic acid exudation rates, and not by zinc-transporter activity

Zinc (Zn)-deficient soils constrain rice (Oryza sativa) production and cause Zn malnutrition. The identification of Zn-deficiency-tolerant rice lines indicates that breeding might overcome these constraints. Here, we seek to identify processes underlying Zn-deficiency tolerance in rice at the physiological and transcriptional levels. A Zn-deficiency-tolerant line RIL46 acquires Zn more efficiently and produces more biomass than its nontolerant maternal line (IR74) at low Zn(ext) under field conditions. We tested if this was the result of increased expression of Zn(2+) transporters; increased root exudation of deoxymugineic acid (DMA) or low-molecular-weight organic acids (LMWOAs); and/or increased root production. Experiments were performed in field and controlled environment conditions. There was little genotypic variation in transcript abundance of Zn-responsive root Zn(2+)-transporters between the RIL46 and IR74. However, root exudation of DMA and LMWOA was greater in RIL46, coinciding with increased root expression of putative ligand-efflux genes. Adventitious root production was maintained in RIL46 at low Zn(ext), correlating with altered expression of root-specific auxin-responsive genes. Zinc-deficiency tolerance in RIL46 is most likely the result of maintenance of root growth, increased efflux of Zn ligands, and increased uptake of Zn-ligand complexes at low Zn(ext); these traits are potential breeding targets.