Development and application of a method for the purification of free shigatoxigenic bacteriophage from environmental samples

Shiga toxin producing Escherichia coli (STEC) strains are foodborne pathogens whose ability to produce Shiga toxin (Stx) is due to the integration of Stx-encoding lambdoid bacteriophage (Stx phage). Circulating, infective Stx phages are very difficult to isolate, purify and propagate such that there is no information on their genetic composition and properties. Here we describe a novel approach that exploits the phage's ability to infect their host and form a lysogen, thus enabling purification of Stx phages by a series of sequential lysogen isolation and induction steps. A total of 15 Stx phages were rigorously purified from water samples in this way, classified by TEM and genotyped using a PCR-based multi-loci characterisation system. Each phage possessed only one variant of each target gene type, thus confirming its purity, with 9 of the 15 phages possessing a short tail-spike gene and identified by TEM as Podoviridae. The remaining 6 phages possessed long tails, four of which appeared to be contractile in nature (Myoviridae) and two of which were morphologically very similar to bacteriophage lambda (Siphoviridae).

Sequence analysis and distribution in Salmonella enterica serovars of IS3-like elements

The genome of Salmonella enterica serovar Enteritidis was shown to possess three IS3-like insertion elements, designated IS1230A, B and C, and each was cloned and their respective deoxynucleotide sequences determined. Mutations in elements IS1230A and B resulted in frameshifts in the open reading frames that encoded a putative transposase to be inactive. IS1230C was truncated at nucleotide 774 relative to IS1230B and therefore did not possess the 3' terminal inverted repeat. The three IS1230 derivatives were closely related to each other based on nucleotide sequence similarity. IS1230A was located adjacent to the sef operon encoding SEF14 fimbriae located at minute 97 of the genome of S. Enteritidis. IS1230B was located adjacent to the umuDC operon at minute 42.5 on the genome, itself located near to one terminus of an 815-kb genome inversion of S. Enteritidis relative to S. Typhimurium. IS1230C was located next to attB, the bacteriophage P22 attachment site, and proB, encoding gamma-glutamyl phosphate reductase. A truncated 3' remnant of IS1230, designated IS1230T, was identified in a clinical isolate of S. Typhimurium DT193 strain 2391. This element was located next to attB adjacent to which were bacteriophage P22-like sequences. Southern hybridisation of total genomic DNA from eighteen phage types of S. Enteritidis and eighteen definitive types of S. Typhimurium showed similar, if not identical, restriction fragment profiles in the respective serovars when probed with IS1230A.

A comparison of Shiga-toxin negative Escherichia coli O157 aflagellate and intimin deficient mutants in porcine in vitro and in vivo models of infection

Isolation of Shiga-toxin (Stx) positive Escherichia coli O157:H7 from commercially grown pigs has been reported. Furthermore, experimental infection studies have demonstrated that Stx-positive E. coli O157:H7 can persist in 12-week-old experimentally orally inoculated conventional pigs for up to 2 months and that persistence was not dependent upon intimin. We have shown that the flagellum of Stx-negative E. coli O157:H7 does not have a role to play in pathogenesis in ruminant models whereas, in poultry, the flagellum of E. coli O157:H7 was important for long-term persistent infection. The contribution of the flagellum of Stx-negative E. coli O157 in the colonisation of pigs was investigated by adherence assays on a porcine (IPI-21) cell line, porcine in vitro organ culture (IVOC) and experimental oral inoculation of conventional 14-week-old pigs. E. coli O157:H7 NCTC12900nal(r) and isogenic aflagellate and intimin deficient mutants adhered equally well to IPI-21 cells. In porcine IVOC association assays, E. coli O157:H7 NCTC12900nal(r) was associated in significantly higher numbers to tissues from the caecum and the terminal rectum than other sites. The aflagellate and intimin deficient mutants significantly adhered in greater numbers to more IVOC gastrointestinal tissues than the parent. Groups of 14-week-old pigs were dosed orally with 10(10) CFU/10 ml of either E. coli O157:H7 NCTC12900nal(r) or isogenic aflagellate and intimin deficient mutants and recovery of each test strain was similar. Histological analysis of pig tissues at post mortem examination revealed that E. coli O157 specifically stained bacteria were associated with the mucosa of the ascending and spiral colon. These data suggest that colonisation and persistence of Stx-negative E. coli O157:H7 in pigs, involves mechanisms that do not require the flagellum or intimin.

Fingerprints of changes in annual and seasonal precipitation from CMIP5 models over land and ocean

By comparing annual and seasonal changes in precipitation over land and ocean since 1950 simulated by the CMIP5 (Coupled Model Intercomparison Project, phase 5) climate models in which natural and anthropogenic forcings have been included, we find that clear global-scale and regional-scale changes due to human influence are expected to have occurred over both land and ocean. These include moistening over northern high latitude land and ocean throughout all seasons and over the northern subtropical oceans during boreal winter. However we show that this signal of human influence is less distinct when considered over the relatively small area of land for which there are adequate observations to make assessments of multi-decadal scale trends. These results imply that extensive and significant changes in precipitation over the land and ocean may have already happened, even though, inadequacies in observations in some parts of the world make it difficult to identify conclusively such a human fingerprint on the global water cycle. In some regions and seasons, due to aliasing of different kinds of variability as a result of sub sampling by the sparse and changing observational coverage, observed trends appear to have been increased, underscoring the difficulties of interpreting the apparent magnitude of observed changes in precipitation.

A comparison of the anticancer properties of isoxanthohumol and 8-prenylnaringenin using in vitro models of colon cancer

The hops plant (Humulus lupulus L.) is an essential ingredient in beer and contains a number of potentially bioactive prenylflavonoids, the predominant being the weakly estrogenic isoxanthohumol (Ix), which can be converted to the more strongly estrogenic 8-PN by the colonic microbiota. The aim of this study was to investigate the biological activity of 8-PN and Ix using in vitro models representing key stages of colorectal carcinogenesis, namely cell growth and viability (MTT assay), cell-cycle progression (DNA content assay), DNA damage (Comet assay), and invasion (Matrigel assay). A significant decrease in Caco-2 cell viability was noted after both 8-PN and Ix treatments at the higher doses (40 and 50 μM, respectively) although the impact on cell cycle differed between the two compounds. The decreased cell viability observed after Ix treatment was associated with a concentration-dependent increase in G2/M and an increased sub-G1 cell-cycle fraction, whereas treatment with 8-PN was associated with an elevated G0/G1 and an increased sub-G1 cell-cycle fraction. Significant antigenotoxic activity was noted at all 8-PN concentrations tested (5-40 μM). Although significant antigenotoxic activity was also noted with Ix treatment at ≤25 μM, at a higher dose, Ix itself exerted genotoxic activity. In a dose-dependent manner, both compounds inhibited HT115 cell invasion with reductions up to 52 and 46% for Ix and 8-PN, respectively, in comparison to untreated cells. This study demonstrated that both Ix and its gut microbial metabolite 8-PN exert anticancer effects on models of key stages of colon tumourigenesis.

Seed development and maturation in early spring-flowering Galanthus nivalis and Narcissus pseudonarcissus continues post-shedding with little evidence of maturation in planta

Background and Aims: Seeds of the moist temperate woodland species Galanthus nivalis and Narcissus pseudonarcissus, dispersed during spring or early summer, germinated poorly in laboratory tests. Seed development and maturation were studied to better understand the progression from developmental to germinable mode in order to improve seed collection and germination practices in these and similar species. Methods: Phenology, seed mass, moisture content, and ability to germinate and tolerate desiccation were monitored during seed development until shedding. Embryo elongation within seeds was investigated during seed development and at several temperature regimes after shedding. Key Results: Seeds were shed at high moisture content (> 59%) with little evidence that dry mass accumulation or embryo elongation were complete. Ability to germinate developed prior to the ability of some seeds to tolerate enforced desiccation. Germination was sporadic and slow. Embryo elongation occurred post-shedding in moist environments, most rapidly at 20C in G. nivalis and 15C in N. pseudonarcissus. The greatest germination also occurred in these regimes, 78 and 48%, respectively, after 700 d. Conclusions: Seeds of G. nivalis and N. pseudonarcissus seeds were comparatively immature at shedding and substantial embryo elongation occurred post-shedding. Seeds showed limited desiccation tolerance at dispersal.

Multi-year droughts in Europe: analysis of development and causes

Whilst hydrological systems can show resilience to short-term streamflow deficiencies during within-year droughts, prolonged deficits during multi-year droughts are a significant threat to water resources security in Europe. This study uses a threshold-based objective classification of regional hydrological drought to qualitatively examine the characteristics, spatio-temporal evolution and synoptic climatic drivers of multi-year drought events in 1962–64, 1975–76 and 1995–97, on a European scale but with particular focus on the UK. Whilst all three events are multi-year, pan-European phenomena, their development and causes can be contrasted. The critical factor in explaining the unprecedented severity of the 1975–76 event is the consecutive occurrence of winter and summer drought. In contrast, 1962–64 was a succession of dry winters, mitigated by quiescent summers, whilst 1995–97 lacked spatial coherence and was interrupted by wet interludes. Synoptic climatic conditions vary within and between multi-year droughts, suggesting that regional factors modulate the climate signal in streamflow drought occurrence. Despite being underpinned by qualitatively similar climatic conditions and commonalities in evolution and characteristics, each of the three droughts has a unique spatio-temporal signature. An improved understanding of the spatio-temporal evolution and characteristics of multi-year droughts has much to contribute to monitoring and forecasting capability, and to improved mitigation strategies.

Uncertainties and assessments of chemistry-climate models of the stratosphere

In recent years a number of chemistry-climate models have been developed with an emphasis on the stratosphere. Such models cover a wide range of time scales of integration and vary considerably in complexity. The results of specific diagnostics are here analysed to examine the differences amongst individual models and observations, to assess the consistency of model predictions, with a particular focus on polar ozone. For example, many models indicate a significant cold bias in high latitudes, the “cold pole problem”, particularly in the southern hemisphere during winter and spring. This is related to wave propagation from the troposphere which can be improved by improving model horizontal resolution and with the use of non-orographic gravity wave drag. As a result of the widely differing modelled polar temperatures, different amounts of polar stratospheric clouds are simulated which in turn result in varying ozone values in the models. The results are also compared to determine the possible future behaviour of ozone, with an emphasis on the polar regions and mid-latitudes. All models predict eventual ozone recovery, but give a range of results concerning its timing and extent. Differences in the simulation of gravity waves and planetary waves as well as model resolution are likely major sources of uncertainty for this issue. In the Antarctic, the ozone hole has probably reached almost its deepest although the vertical and horizontal extent of depletion may increase slightly further over the next few years. According to the model results, Antarctic ozone recovery could begin any year within the range 2001 to 2008. The limited number of models which have been integrated sufficiently far indicate that full recovery of ozone to 1980 levels may not occur in the Antarctic until about the year 2050. For the Arctic, most models indicate that small ozone losses may continue for a few more years and that recovery could begin any year within the range 2004 to 2019. The start of ozone recovery in the Arctic is therefore expected to appear later than in the Antarctic.

Historical and idealized climate model experiments: an intercomparison of Earth system models of intermediate complexity

Both historical and idealized climate model experiments are performed with a variety of Earth system models of intermediate complexity (EMICs) as part of a community contribution to the Intergovernmental Panel on Climate Change Fifth Assessment Report. Historical simulations start at 850 CE and continue through to 2005. The standard simulations include changes in forcing from solar luminosity, Earth's orbital configuration, CO2, additional greenhouse gases, land use, and sulphate and volcanic aerosols. In spite of very different modelled pre-industrial global surface air temperatures, overall 20th century trends in surface air temperature and carbon uptake are reasonably well simulated when compared to observed trends. Land carbon fluxes show much more variation between models than ocean carbon fluxes, and recent land fluxes appear to be slightly underestimated. It is possible that recent modelled climate trends or climate–carbon feedbacks are overestimated resulting in too much land carbon loss or that carbon uptake due to CO2 and/or nitrogen fertilization is underestimated. Several one thousand year long, idealized, 2 × and 4 × CO2 experiments are used to quantify standard model characteristics, including transient and equilibrium climate sensitivities, and climate–carbon feedbacks. The values from EMICs generally fall within the range given by general circulation models. Seven additional historical simulations, each including a single specified forcing, are used to assess the contributions of different climate forcings to the overall climate and carbon cycle response. The response of surface air temperature is the linear sum of the individual forcings, while the carbon cycle response shows a non-linear interaction between land-use change and CO2 forcings for some models. Finally, the preindustrial portions of the last millennium simulations are used to assess historical model carbon-climate feedbacks. Given the specified forcing, there is a tendency for the EMICs to underestimate the drop in surface air temperature and CO2 between the Medieval Climate Anomaly and the Little Ice Age estimated from palaeoclimate reconstructions. This in turn could be a result of unforced variability within the climate system, uncertainty in the reconstructions of temperature and CO2, errors in the reconstructions of forcing used to drive the models, or the incomplete representation of certain processes within the models. Given the forcing datasets used in this study, the models calculate significant land-use emissions over the pre-industrial period. This implies that land-use emissions might need to be taken into account, when making estimates of climate–carbon feedbacks from palaeoclimate reconstructions.

Theory and generalization of Monte Carlo models of the BOLD signal source

The dependency of the blood oxygenation level dependent (BOLD) signal on underlying hemodynamics is not well understood. Building a forward biophysical model of this relationship is important for the quantitative estimation of the hemodynamic changes and neural activity underlying functional magnetic resonance imaging (fMRI) signals. We have developed a general model of the BOLD signal which can model both intra- and extravascular signals for an arbitrary tissue model across a wide range of imaging parameters. The model of the BOLD signal was instantiated as a look-up-table (LuT), and was verified against concurrent fMRI and optical imaging measurements of activation induced hemodynamics. Magn Reson Med, 2008. © 2008 Wiley-Liss, Inc.

A SEPALLATA gene is involved in the development and ripening of strawberry (Fragaria X ananassa Duch.) fruit, a non-climacteric tissue

Climacteric and non-climacteric fruits have traditionally been viewed as representing two distinct programmes of ripening associated with differential respiration and ethylene hormone effects. In climacteric fruits, such as tomato and banana, the ripening process is marked by increased respiration and is induced and co-ordinated by ethylene, while in non-climacteric fruits, such as strawberry and grape, it is controlled by an ethylene-independent process with little change in respiration rate. The two contrasting mechanisms, however, both lead to texture, colour, and flavour changes that probably reflect some common programmes of regulatory control. It has been shown that a SEPALLATA(SEP)4-like gene is necessary for normal ripening in tomato. It has been demonstrated here that silencing a fruit-related SEP1/2-like (FaMADS9) gene in strawberry leads to the inhibition of normal development and ripening in the petal, achene, and receptacle tissues. In addition, analysis of transcriptome profiles reveals pleiotropic effects of FaMADS9 on fruit development and ripening-related gene expression. It is concluded that SEP genes play a central role in the developmental regulation of ripening in both climacteric and non-climacteric fruits. These findings provide important information to extend the molecular control of ripening in a non-climacteric fruit beyond the limited genetic and cultural options currently available.

Development and evaluation of an Earth-System model – HadGEM2

We describe here the development and evaluation of an Earth system model suitable for centennial-scale climate prediction. The principal new components added to the physical climate model are the terrestrial and ocean ecosystems and gas-phase tropospheric chemistry, along with their coupled interactions. The individual Earth system components are described briefly and the relevant interactions between the components are explained. Because the multiple interactions could lead to unstable feedbacks, we go through a careful process of model spin up to ensure that all components are stable and the interactions balanced. This spun-up configuration is evaluated against observed data for the Earth system components and is generally found to perform very satisfactorily. The reason for the evaluation phase is that the model is to be used for the core climate simulations carried out by the Met Office Hadley Centre for the Coupled Model Intercomparison Project (CMIP5), so it is essential that addition of the extra complexity does not detract substantially from its climate performance. Localised changes in some specific meteorological variables can be identified, but the impacts on the overall simulation of present day climate are slight. This model is proving valuable both for climate predictions, and for investigating the strengths of biogeochemical feedbacks.

Transcriptional dysregulation of coding and non-coding genes in cellular models of Huntington's disease

HD (Huntington's disease) is a late onset heritable neurodegenerative disorder that is characterized by neuronal dysfunction and death, particularly in the cerebral cortex and medium spiny neurons of the striatum. This is followed by progressive chorea, dementia and emotional dysfunction, eventually resulting in death. HD is caused by an expanded CAG repeat in the first exon of the HD gene that results in an abnormally elongated polyQ (polyglutamine) tract in its protein product, Htt (Huntingtin). Wild-type Htt is largely cytoplasmic; however, in HD, proteolytic N-terminal fragments of Htt form insoluble deposits in both the cytoplasm and nucleus, provoking the idea that mutHtt (mutant Htt) causes transcriptional dysfunction. While a number of specific transcription factors and co-factors have been proposed as mediators of mutHtt toxicity, the causal relationship between these Htt/transcription factor interactions and HD pathology remains unknown. Previous work has highlighted REST [RE1 (repressor element 1)-silencing transcription factor] as one such transcription factor. REST is a master regulator of neuronal genes, repressing their expression. Many of its direct target genes are known or suspected to have a role in HD pathogenesis, including BDNF (brain-derived neurotrophic factor). Recent evidence has also shown that REST regulates transcription of regulatory miRNAs (microRNAs), many of which are known to regulate neuronal gene expression and are dysregulated in HD. Thus repression of miRNAs constitutes a second, indirect mechanism by which REST can alter the neuronal transcriptome in HD. We will describe the evidence that disruption to the REST regulon brought about by a loss of interaction between REST and mutHtt may be a key contributory factor in the widespread dysregulation of gene expression in HD.