Sustained polymeric delivery of gene silencing antisense ODNs, siRNA, DNAzymes and ribozymes: in vitro and in vivo studies

Small interfering RNA (siRNA), antisense oligonucleotides (ODNs), ribozymes and DNAzymes have emerged as sequence-specific inhibitors of gene expression that may have therapeutic potential in the treatment of a wide range of diseases. Due to their rapid degradation in vivo, the efficacy of naked gene silencing nucleic acids is relatively short lived. The entrapment of these nucleic acids within biodegradable sustained-release delivery systems may improve their stability and reduce the doses required for efficacy. In this study, we have evaluated the potential in vitro and in vivo use of biodegradable poly (d,l-lactide-co-glycolide) copolymer (PLGA) microspheres as sustained delivery devices for ODNs, ribozyme, siRNA and DNA enzymes. In addition, we investigated the release of ODN conjugates bearing 5′-end lipophilic groups. The in vitro sustained release profiles of microsphere-entrapped nucleic acids were dependent on variables such as the type of nucleic acid used, the nature of the lipophilic group, and whether the nucleic acid used was single or double stranded. For in vivo studies, whole body autoradiography was used to monitor the bio-distribution of either free tritium-labelled ODN or that entrapped within PLGA microspheres following subcutaneous administration in Balb-c mice. The majority of the radioactivity associated with free ODN was eliminated within 24 h whereas polymer-released ODN persisted in organs and at the site of administration even after seven days post-administration. Polymer microsphere released ODN exhibited a similar tissue and cellular tropism to the free ODN. Micro-autoradiography analyses of the liver and kidneys showed similar bio-distribution for polymer-released and free ODNs with the majority of radioactivity being concentrated in the proximal convoluted tubules of the kidney and in the Kupffer cells of the liver. These findings suggest that biodegradable PLGA microspheres offer a method for improving the in vivo sustained delivery of gene silencing nucleic acids, and hence are worthy of further investigation as delivery systems for these macromolecules.

The Climate-G testbed: towards large scale distributed data management for climate change

Climate-G is a large scale distributed testbed devoted to climate change research. It is an unfunded effort started in 2008 and involving a wide community both in Europe and US. The testbed is an interdisciplinary effort involving partners from several institutions and joining expertise in the field of climate change and computational science. Its main goal is to allow scientists carrying out geographical and cross-institutional data discovery, access, analysis, visualization and sharing of climate data. It represents an attempt to address, in a real environment, challenging data and metadata management issues. This paper presents a complete overview about the Climate-G testbed highlighting the most important results that have been achieved since the beginning of this project.

The REFLEX project: Comparing different algorithms and implementations for the inversion of a terrestrial ecosystem model against eddy covariance data

We describe a model-data fusion (MDF) inter-comparison project (REFLEX), which compared various algorithms for estimating carbon (C) model parameters consistent with both measured carbon fluxes and states and a simple C model. Participants were provided with the model and with both synthetic net ecosystem exchange (NEE) of CO2 and leaf area index (LAI) data, generated from the model with added noise, and observed NEE and LAI data from two eddy covariance sites. Participants endeavoured to estimate model parameters and states consistent with the model for all cases over the two years for which data were provided, and generate predictions for one additional year without observations. Nine participants contributed results using Metropolis algorithms, Kalman filters and a genetic algorithm. For the synthetic data case, parameter estimates compared well with the true values. The results of the analyses indicated that parameters linked directly to gross primary production (GPP) and ecosystem respiration, such as those related to foliage allocation and turnover, or temperature sensitivity of heterotrophic respiration, were best constrained and characterised. Poorly estimated parameters were those related to the allocation to and turnover of fine root/wood pools. Estimates of confidence intervals varied among algorithms, but several algorithms successfully located the true values of annual fluxes from synthetic experiments within relatively narrow 90% confidence intervals, achieving >80% success rate and mean NEE confidence intervals <110 gC m−2 year−1 for the synthetic case. Annual C flux estimates generated by participants generally agreed with gap-filling approaches using half-hourly data. The estimation of ecosystem respiration and GPP through MDF agreed well with outputs from partitioning studies using half-hourly data. Confidence limits on annual NEE increased by an average of 88% in the prediction year compared to the previous year, when data were available. Confidence intervals on annual NEE increased by 30% when observed data were used instead of synthetic data, reflecting and quantifying the addition of model error. Finally, our analyses indicated that incorporating additional constraints, using data on C pools (wood, soil and fine roots) would help to reduce uncertainties for model parameters poorly served by eddy covariance data.

Inducible hydrogen sulfide synthesis in chondrocytes and mesenchymal progenitor cells: is H2S a novel cytoprotective mediator in the inflamed joint?

Hydrogen sulfide (H(2)S) has recently been proposed as an endogenous mediator of inflammation and is present in human synovial fluid. This study determined whether primary human articular chondrocytes (HACs) and mesenchymal progenitor cells (MPCs) could synthesize H(2)S in response to pro-inflammatory cytokines relevant to human arthropathies, and to determine the cellular responses to endogenous and pharmacological H(2)S. HACs and MPCs were exposed to IL-1β, IL-6, TNF-α and lipopolysaccharide (LPS). The expression and enzymatic activity of the H(2)S synthesizing enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) were determined by Western blot and zinc-trap spectrophotometry, respectively. Cellular oxidative stress was induced by H(2)O(2), the peroxynitrite donor SIN-1 and 4-hydroxynonenal (4-HNE). Cell death was assessed by 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Mitochondrial membrane potential (DCm) was determined in situ by flow cytometry. Endogenous H(2) S synthesis was inhibited by siRNA-mediated knockdown of CSE and CBS and pharmacological inhibitors D,L-propargylglycine and aminoxyacetate, respectively. Exogenous H(2)S was generated using GYY4137. Under basal conditions HACs and MPCs expressed CBS and CSE and synthesized H(2)S in a CBS-dependent manner, whereas CSE expression and activity was induced by treatment of cells with IL-1β, TNF-α, IL-6 or LPS. Oxidative stress-induced cell death was significantly inhibited by GYY4137 treatment but increased by pharmacological inhibition of H(2)S synthesis or by CBS/CSE-siRNA treatment. These data suggest CSE is an inducible source of H(2)S in cultured HACs and MPCs. H(2)S may represent a novel endogenous mechanism of cytoprotection in the inflamed joint, suggesting a potential opportunity for therapeutic intervention.

A global perspective on Langmuir turbulence in the ocean surface boundary layer.

The turbulent mixing in thin ocean surface boundary layers (OSBL), which occupy the upper 100 m or so of the ocean, control the exchange of heat and trace gases between the atmosphere and ocean. Here we show that current parameterizations of this turbulent mixing lead to systematic and substantial errors in the depth of the OSBL in global climate models, which then leads to biases in sea surface temperature. One reason, we argue, is that current parameterizations are missing key surface-wave processes that force Langmuir turbulence that deepens the OSBL more rapidly than steady wind forcing. Scaling arguments are presented to identify two dimensionless parameters that measure the importance of wave forcing against wind forcing, and against buoyancy forcing. A global perspective on the occurrence of waveforced turbulence is developed using re-analysis data to compute these parameters globally. The diagnostic study developed here suggests that turbulent energy available for mixing the OSBL is under-estimated without forcing by surface waves. Wave-forcing and hence Langmuir turbulence could be important over wide areas of the ocean and in all seasons in the Southern Ocean. We conclude that surfacewave- forced Langmuir turbulence is an important process in the OSBL that requires parameterization.

Behavioral and spatial analysis of extraterritorial movements in red foxes (Vulpes vulpes)

Abstract: Movements away from the natal or home territory are important to many ecological processes, including gene flow, population regulation, and disease epidemiology, yet quantitative data on these behaviors are lacking. Red foxes exhibit 2 periods of extraterritorial movements: when an individual disperses and when males search neighboring territories for extrapair copulations during the breeding season. Using radiotracking data collected at 5-min interfix intervals, we compared movement parameters, including distance moved, speed of movement, and turning angles, of dispersal and reproductive movements to those made during normal territorial movements; the instantaneous separation distances of dispersing and extraterritorial movements to the movements of resident adults; and the frequency of locations of 95%, 60%, and 30% harmonic mean isopleths of adult fox home territories to randomly generated fox movements. Foxes making reproductive movements traveled farther than when undertaking other types of movement, and dispersal movements were straighter. Reproductive and dispersal movements were faster than territorial movements and also differed in intensity of search and thoroughness. Foxes making dispersal movements avoided direct contact with territorial adults and moved through peripheral areas of territories. The converse was true for reproductive movements. Although similar in some basic characteristics, dispersal and reproductive movements are fundamentally different both behaviorally and spatially and are likely to have different ultimate purposes and contrasting effects on spatial processes such as disease transmission

Storytelling in song: television music, narrative and allusion in 'The O.C.'

Television’s long-form storytelling has the potential to allow the rippling of music across episodes and seasons in interesting ways. In the integration of narrative, music and meaning found in The O.C. (Fox, FOX 2003-7), popular song’s allusive and referential qualities are drawn upon to particularly televisual ends. At times embracing its ‘disruptive’ presence, at others suturing popular music into narrative, at times doing both at once. With television studies largely lacking theories of music, this chapter draws on film music theory and close textual analysis to analyse some of the programme's music moments in detail. In particular it considers the series-spanning use of Jeff Buckley’s cover of ‘Hallelujah’ (and its subsequent oppressive presence across multiple televisual texts), the end of episode musical montage and the use of recurring song fragments as theme within single episodes. In doing so it highlights music's role in the fragmentation and flow of the television aesthetic and popular song’s structural presence in television narrative. Illustrating the multiplicity of popular song’s use in television, these moments demonstrate song’s ability to provide narrative commentary, yet also make particular use of what Ian Garwood describes as the ability of ‘a non-diegetic song to exceed the emotional range displayed by diegetic characters’ (2003:115), to ‘speak’ for characters or to their feelings, contributing to both teen TV’s melodramatic affect and narrative expression.

Species richness declines and biotic homogenization have slowed down for NW-European pollinators and plants

Concern about biodiversity loss has led to increased public investment in conservation. Whereas there is a widespread perception that such initiatives have been unsuccessful, there are few quantitative tests of this perception. Here, we evaluate whether rates of biodiversity change have altered in recent decades in three European countries (Great Britain, Netherlands and Belgium) for plants and flower visiting insects. We compared four 20-year periods, comparing periods of rapid land-use intensification and natural habitat loss (1930–1990) with a period of increased conservation investment (post-1990). We found that extensive species richness loss and biotic homogenisation occurred before 1990, whereas these negative trends became substantially less accentuated during recent decades, being partially reversed for certain taxa (e.g. bees in Great Britain and Netherlands). These results highlight the potential to maintain or even restore current species assemblages (which despite past extinctions are still of great conservation value), at least in regions where large-scale land-use intensification and natural habitat loss has ceased.

Infrared Observations of XTE J1118+480

Infrared observations of the outbursting black hole XTE J1118+480 (ATEL #383) were performed using SQIID on the Kitt Peak National Observatory 2.1m telescope. Observations spanning 2005 January 15.42-15.58 found it somewhat fainter than the previous outburst (IAUC # 7394 , # 7407 ), at average brightness J=12.91+/-0.03, H=12.50+/-0.03, K=11.95+/-0.03. The colors again correspond to an approximately flat spectrum in F_nu. No orbital variation is apparent, but there is substantial unresolved rapid variability with rms amplitude 22% in K (between 2s exposures). Further observations are planned nightly until Jan 21.

“There’s something very familiar about all this”: generic play and performance in the Back to the Future trilogy (Robert Zemeckis, 1985, 1989, 1990)

The Back to the Future Trilogy incorporates several different generic elements, including aspects of the fifties teen movie, science fiction, comedy and the western. These different modes playfully intertwine with each other creating a complex world of repetitions, echoes and modulations. This essay seeks to interrogate the construction of generic elements and the play between them through a close analysis of a repeated performance. Genre is signalled through various strategies employed within the construction of mise-en-scène, a significant portion of this, as I would like to argue, is transmitted through performance. The material detail of a performance – incorporating gesture, movement, voice, and even surrounding elements such as costume – as well as the way it its presented within a film is key to the establishment, invocation and coherence of genre. Furthermore, attention to the complexity of performance details, particularly in the manner in which they reverberate across texts, demonstrates the intricacy of genre and its inherent mutability. The Back to the Future trilogy represents a specific interest in the flexibility of genre. Within each film, and especially across all three, aspects of various genres are interlaced through both visual and narrative detail, thus constructing a dense layer of references both within and without the texts. To explore this patterning in more detail I will interrogate the contribution of performance to generic play through close analysis of Thomas F. Wilson’s performance of Biff/Griff/Burford Tannen and his central encounter with Marty McFly (Michael J. Fox) in each film. These moments take place in a fifties diner, a 1980s retro diner and a saloon respectively, each space contributing the similarities and differences in each repetition. Close attention to Wilson’s performance of each related character, which contains both modulations and repetitions used specifically to place each film’s central generic theme, demonstrates how embedded the play between genres and their flexibility is within the trilogy.

Array based detection of antibiotic resistance genes in Gram negative bacteria isolated from retail poultry meat in the UK and Ireland

The use of antibiotics in birds and animals intended for human consumption within the European Union (EU) and elsewhere has been subject to regulation prohibiting the use of antimicrobials as growth promoters and the use of last resort antibiotics in an attempt to reduce the spread of multi-resistant Gram negative bacteria. Given the inexorable spread of antibiotic resistance there is an increasing need for improved monitoring of our food. Using selective media, Gram negative bacteria were isolated from retail chicken of UK-Intensively reared (n = 27), Irish-Intensively reared (n = 19) and UK-Free range (n = 30) origin and subjected to an oligonucleotide based array system for the detection of 47 clinically relevant antibiotic resistance genes (ARGs) and two integrase genes. High incidences of β-lactamase genes were noted in all sample types, acc (67%), cmy (80%), fox (55%) and tem (40%) while chloramphenicol resistant determinants were detected in bacteria from the UK poultry portions and were absent in bacteria from the Irish samples. Denaturing Gradient Gel Electrophoresis (DGGE) was used to qualitatively analyse the Gram negative population in the samples and showed the expected diversity based on band stabbing and DNA sequencing. The array system proved to be a quick method for the detection of antibiotic resistance gene (ARG) burden within a mixed Gram negative bacterial population.

Changes in the distribution of red foxes (Vulpes vulpes) in urban areas in Great Britain: findings and limitations of a media-driven nationwide survey

Urbanization is one of the major forms of habitat alteration occurring at the present time. Although this is typically deleterious to biodiversity, some species flourish within these human-modified landscapes, potentially leading to negative and/or positive interactions between people and wildlife. Hence, up-to-date assessment of urban wildlife populations is important for developing appropriate management strategies. Surveying urban wildlife is limited by land partition and private ownership, rendering many common survey techniques difficult. Garnering public involvement is one solution, but this method is constrained by the inherent biases of non-standardised survey effort associated with voluntary participation. We used a television-led media approach to solicit national participation in an online sightings survey to investigate changes in the distribution of urban foxes in Great Britain and to explore relationships between urban features and fox occurrence and sightings density. Our results show that media-based approaches can generate a large national database on the current distribution of a recognisable species. Fox distribution in England and Wales has changed markedly within the last 25 years, with sightings submitted from 91% of urban areas previously predicted to support few or no foxes. Data were highly skewed with 90% of urban areas having <30 fox sightings per 1000 people km-2. The extent of total urban area was the only variable with a significant impact on both fox occurrence and sightings density in urban areas; longitude and percentage of public green urban space were respectively, significantly positively and negatively associated with sightings density only. Latitude, and distance to nearest neighbouring conurbation had no impact on either occurrence or sightings density. Given the limitations associated with this method, further investigations are needed to determine the association between sightings density and actual fox density, and variability of fox density within and between urban areas in Britain.

Ionospheric ion and electron heating at the poleward boundary of a poleward expanding substorm-disturbed region

We present observations of a poleward propagating substorm-disturbed region which was observed by the European Incoherent SCATter (EISCAT) radar and the Svalbard International Monitor for Auroral Geomagnetic Effects (IMAGE) magnetometers in the postmidnight sector. The expansion of the disturbance was launched by a substorm intensification which started similar to 25 min after the initial onset, and similar to 10 min before the disturbance arrived over Svalbard. In association with the magnetic disturbance, a poleward expanding enduring enhancement in the F region electron temperature was observed, indicative of soft electron precipitation, with a narrow band of enhanced ion temperature straddling its poleward edge, indicative of fast ion flows and ion-neutral collisional heating. This electron temperature boundary was coincident with the poleward propagating electrojet current system detected by the high-latitude IMAGE magnetometer stations and is taken to be a proxy for the observation of a substorm auroral bulge. The electron temperature boundary is inferred to have a width comparable or less than one radar range gate (similar to 60 km transverse to the magnetic field), while the region of high ion temperature was found to be approximately three gates wide, extending approximately two gates (similar to 120 km) poleward of the electron temperature boundary, and approximately one gate (similar to 60 km) equatorward. The two-beam radar line-of-sight velocity data are found to be consistent with the existence of a layer of high-speed flow in the boundary, peaking at values similar to1.5-3 km s(-1), roughly consistent with the ion temperature data. The flow is directed either east or west along the boundary depending on the direction of the flow in the poleward region. We infer that the flow is deflected along and around the boundary of the substorm-disturbed region due to the high conductivity of the latter. Variations in the flow poleward of the boundary produced no discernible magnetic effects on the ground, confirming the low conductivity of the preboundary ionosphere.

A multipoint study of a substorm occurring on 7 December, 1992, and its theoretical implications

On 7 December 1992, a moderate substorm was observed by a variety of satellites and ground-based instruments. Ionospheric flows were monitored near dusk by the Goose Bay HF radar and near midnight by the EISCAT radar. The observed flows are compared here with magnetometer observations by the IMAGE array in Scandinavia and the two Greenland chains, the auroral distribution observed by Freja and the substorm cycle observations by the SABRE radar, the SAMNET magnetometer array and LANL geosynchronous satellites. Data from Galileo Earth-encounter II are used to estimate the IMF B-z component. The data presented show that the substorm onset electrojet at midnight was confined to closed field lines equatorward of the preexisting convection reversal boundaries observed in the dusk and midnight regions. No evidence of substantial closure of open flux was detected following this substorm onset. Indeed the convection reversal boundary on the duskside continued to expand equatorward after onset due to the continued presence of strong southward IMF, such that growth and expansion phase features were simultaneously present. Clear indications of closure of open flux were not observed until a subsequent substorm intensification 25 min after the initial onset. After this time, the substorm auroral bulge in the nightside hours propagated well poleward of the pre-existing convection reversal boundary, and strong flow perturbations were observed by the Goose Bay radar, indicative of flows driven by reconnection in the tail.