Sequence-Dependent Promoter Escape Efficiency Is Strongly Influenced by Bias for the Pretranslocated State during Initial Transcription.

Abortive transcription initiation can be rate-limiting for promoter escape and therefore represents a barrier to productive gene expression. The mechanism for abortive initiation is unknown, but the amount of abortive transcript is known to vary with the composition of the initial transcribed sequence (ITS). Here, we used a thermodynamic model of translocation combined with experimental validation to investigate the relationship between ITS and promoter escape on a set of phage T5 N25 promoters. We found a strong, negative correlation between RNAP's propensity to occupy the pretranslocated state during initial transcription and the efficiency of promoter escape (r = -0.67; p < 10(-6)). This correlation was almost entirely caused by free energy changes due to variation in the RNA 3' dinucleotide sequence at each step, implying that this sequence element controls the disposition of initial transcribing complexes. We tested our model experimentally by constructing a set of novel N25-ITS promoter variants; quantitative transcription analysis again showed a strong correlation (r = -0.81; p < 10(-6)). Our results support a model in which sequence-directed bias for the pretranslocated state during scrunching results in increased backtracking, which limits the efficiency of promoter escape. This provides an answer to the long-standing issue of how sequence composition of the ITS affects promoter escape efficiency.

Design and optimization of short DNA sequences that can be used as 5' fusion partners for high-level expression of heterologous genes in Escherichia coli.

The 5' terminal nucleotide sequence of a gene is often a bottleneck in recombinant protein production. The ifn-α2bS gene is poorly expressed in Escherichia coli unless a translocation signal sequence (pelB) is fused to the 5' end of the gene. A combined in silico and in vivo analysis reported here further indicates that the ifn-α2bS 5' coding sequence is suboptimal for efficient gene expression. ifn-α2bS therefore presents a suitable model gene for describing properties of 5' fusions promoting expression. We show that short DNA sequences corresponding to the 5' end of the highly expressed celB gene, whose protein product is cytosolic, can functionally replace pelB as a 5' fusion partner for efficient ifn-α2bS expression. celB fusions of various lengths (corresponding to a minimum of 8 codons) led to more than 7- and 60-fold stimulation of expression at the transcript and protein levels, respectively. Moreover, the presence of a celB-based fusion partner was found to moderately reduce the decay rate of the corresponding transcript. The 5' fusions thus appear to act by enhancing translation, and bound ribosomes may accordingly contribute to increased mRNA stability and reduced mRNA decay. However, other effects, such as altered protein stability, cannot be excluded. We also developed an experimental protocol that enabled us to identify improved variants of the celB fusion, and one of these (celBD11) could be used to additionally increase ifn-α2bS expression more than 4-fold at the protein level. Interestingly, celBD11 also stimulated greater protein production of three other medically important human genes than the wild-type celB fragment.

Effects of chemical dispersants on feathers from Arctic seabirds.

Chemical dispersion is an oil spill response strategy where dispersants are sprayed onto the oil slick to enhance oil dispersion into the water. However, accidental application could expose seabirds to dispersants, thereby negatively affecting their plumage. To understand the possible impacts on seabirds, feathers from common eider (Somateria mollissima) and thick-billed murre (Uria lomvia) were exposed to different dosages of the dispersant Dasic Slickgone NS. For all exposure dosages the feathers increased in weight, and mostly for common eider. Analysing the feather microstructure, e.g., the Amalgamation Index, showed that larger damages were found on thick-billed murre than common eider. A no-sinking limit was established at 0.109 ml/m2. Relating this value to desktop simulations of potential sea-surface dosages in real-life situations, and to published accounts of response operations, showed that the limit is likely to be exceeded. Thus, our results show that chemical dispersants in realistic dosages could impact seabirds.

North sea produced water PAH exposure and uptake in early life stages of Atlantic Cod.

Produced water discharges from offshore oil and gas platforms represent a significant source of petroleum components such as polycyclic aromatic hydrocarbons (PAHs) released to the ocean. High molecular weight PAHs are persistent in the environment and have a potential for bioaccumulation, and the investigation of their fate and uptake pathways in marine life are relevant when assessing environmental risk of produced water discharges. To study the exposure and uptake of 2-5 ring PAHs in early life stages of Atlantic Cod in the North Sea, we run a coupled fate and individual-based numerical model that includes discharges from 26 platforms. We consider 26 different PAH components in produced water which biodegrade with primary degradation rates; intermediate degradation products are not included. Model simulations are run covering multiple years (2009-2012) to study annual exposure variability, while a one-day time slice of spawning products from the peak spawning season are followed. By covering multiple release points and large spatio-temporal scales, we show how individuals can be exposed to produced water from multiple regions in the North Sea. We find that a combination of oceanic fate processes and toxicokinetics lead to markedly different compositions in the predicted internal concentrations of PAHs compared to discharge concentrations; for instance, naphthalene makes up 30% of the total discharged PAHs, but contributes to at most 1% of internal concentrations. In all simulations we find the predicted total internal PAH concentration (26 components) to be below 1.2 nmol/g, a factor of 1000 less than concentrations commonly associated with acute narcotic effects.

Attachment of APAM to mineral particles in seawater.

Polymer injection is used in enhanced oil recovery (EOR) when an oil field ages and the pressure in the reservoir decreases, or for oil fields with heavy oil. By polymer injection, the viscosity of the water injected for pressure support is increased by mixing with a high concentration of a polymer solution. Polymers used in EOR operations are often high molecular weight polyacrylamides, including anionic polyacrylamide (APAM), which may subsequently enter the marine environment with produced water releases. Since seawater (SW) contains mineral particles (MPs) in low concentrations, and polymers like APAM are known to flocculate MPs, we investigated if APAM at different concentrations (0.5-10 mg/L) would attach and flocculate MPs, when these occurred in concentrations relevant for oceanic SW (1 mg/L). Two types of MPs, diatomaceous earth and kaolin, were exposed to fluorescence-tagged APAM (APAM-TAG). A low-energy carousel system with natural seawater (SW) was used for incubation of MPs and APAM-TAG at a temperature relevant for the Norwegian Continental Shelf (13 °C). Attachment to MPs and aggregates of these were analysed by fluorometry and fluorescence microscopy. Particle analyses showed that only minor fractions of the MPs aggregated. When samples were separated in steel filter with a mesh size of 20 µm, APAM-TAG was mainly measured in the flow-through fraction (<20 µm), and the results therefore showed that the polymer mainly remained in the water-phase, or was attached to small particles (<20 µm). For the small fraction of APAM attaching to aggregated MPs, attraction to kaolin was higher than to diatomaceous earth, and fluorescence microscopy analyses confirmed the presence of fluorescent particles at the higher APAM concentrations. MPs at concentrations relevant for oceanic SW are therefore not expected to significantly contribute to sedimentation of APAM dissolved in the water column.

Modelling of oil thickness in the presence of an ice edge.

Oil slick thickness is a key parameter for the behaviour of oil spilled at sea. It influences evaporation and entrainment, viable response options, and the risk to marine life at the surface. Determining this value is therefore of high relevance in oil spill modelling. In open water, oil can spread as thin films due to gravity alone, and may be further dispersed by horizontal diffusion and differential advection. In the presence of ice, however, a thin oil slick may become concentrated to higher thickness, if compressed against the ice edge. In the present study, we develop a simple model for the thickness of oil forced against a barrier by a current. We compare our theory to flume experiments, and obtain reasonable agreement. We describe an implementation in a Lagrangian oil spill model, and present some examples. We discuss the operational applicability, and suggest further research needs.

The use of PAH, metabolite and lipid profiling to assess exposure and effects of produced water discharges on pelagic copepods.

Several laboratory studies have demonstrated that exposure to oil components cause toxicity to copepods, however, this has never been shown in natural populations of copepods. In the present study, we sampled copepods in an area of the North Sea with high density of oil production platforms discharging produced water. Environmental modelling was used to predict produced water and copepod trajectories prior to copepod sampling in situ. To maximise output from a minimal number of field samples, a novel and combined methodology was developed to allow exploitation of the same extract for several purposes; contaminant body burden, lipidomics, and metabolomics analysis. PAH body burdens were low compared to laboratory experiments where correlations between PAH body burden and acute toxicity, reproduction and molecular endpoints had been established. Still, station-specific PAH profiles strongly indicated copepod exposure to PW. NMR metabolomics, focusing on water-soluble metabolites, suggested no correlation between metabolites and stations. Interestingly, lipidomics analyses suggested site-specific fingerprints and profiles displayed for acyl-glycerols and wax esters. Potential effects of produced water exposure on lipid metabolism in copepods cannot be ruled out and deserves more attention. Our study exemplifies the importance of incorporating novel and improved analytical methodologies in environmental monitoring.

Improving oil spill trajectory modelling in the Arctic.

As petroleum development and other activities move further north, the potential for oil spills in ice-covered waters is of great concern. As a tool for contingency planning and forecasting during response, oil spill models play a key role. With the development of new, high-resolution coupled ice-ocean models, better predictions of sea ice are becoming available. We have updated the OSCAR oil spill model to use sea-ice velocity and coverage fields from coupled ice-ocean models to improve simulation of oil fate and transport in ice-covered waters. We describe the implementation of oil transport in the presence of ice, and demonstrate the improvement by considering three case studies. We find clear improvement when taking ice velocity from a coupled ice-ocean model into account, compared to a heuristic model that uses surface current and wind velocity. The difference is found to be especially important in a response situation near the marginal ice zone.

Acute and sub-lethal effects of an anionic polyacrylamide on sensitive early life stages of Atlantic cod (Gadus morhua).

Despite the possible increase in use of anionic polyacrylamide (APAM) in enhanced oil recovery operations, very little relevant information regarding ecotoxicity exists. The current study assessed acute and sub-lethal toxicity in sensitive early life stages (ELS) of Atlantic cod (Gadus morhua) exposed to 200 kDa APAM under controlled laboratory conditions. Two experiments (screening and long-term study) were conducted covering ecologically relevant endpoints (survival, hatching, growth, deformations, respiration and heart rate) in fish developing through embryogenesis, hatching, yolk-sac larvae stage and the first feeding period. The screening experiment was an 8-day exposure of embryos, whereas in the long-term experiments embryos and developing larvae were exposed continuously for 23 days. In the screening experiment, a significant reduction in embryonic heart rate was observed during exposure to 150 and 1500 mg APAM/L. However, we observed no effects on fitness-related endpoints (survival, hatching and growth) at concentrations up to 1500 mg L-1 APAM. Also, for the long-term exposure from late embryo to first feeding larvae stage, we observed reduced heart rate at 125 mg L-1. No consistent responses on survival, growth or respiration were observed except for the highest concentration tested (6000 mg L-1). Dispersion modelling based on expected and relevant discharged polymer concentrations and durations showed that predicted environmental concentrations were orders of magnitude lower than the concentrations tested in our experiments, indicating that 200 kDa APAM will have a limited probability of causing fitness-related effects on Atlantic cod ELS.

The sensitivity of the surface oil signature to subsurface dispersant injection and weather conditions.

Subsea blowouts have the potential to spread oil across large geographical areas, and subsea dispersant injection (SSDI) is a response option targeted at reducing the impact of a blowout, especially reducing persistent surface oil slicks. Modified Weber scaling was used to predict oil droplet sizes with the OSCAR oil spill model, and to evaluate the surface oil volume and area when using SSDI under different conditions. Generally, SSDI reduces the amount of oil on the surface, and creates wider and thinner surface oil slicks. It was found that the reduction of surface oil area and volume with SSDI was enhanced for higher wind speeds. Overall, given the effect of SSDI on oil volume and weathering, it may be suggested that tar ball formation, requiring thick and weathered oil, could possibly be reduced when SSDI is used.

Management of oil spill contamination in the Gulf of Patras caused by an accidental subsea blowout.

A methodology is presented and applied to assess the oil contamination probability in the Gulf of Patras and the environmental impacts on the environmentally sensitive area of Mesolongi - Aitoliko coastal lagoons, and to examine the effectiveness of response systems. The procedure consists of the following steps: (1) Determination of the computational domain and the main areas of interest, (2) determination of the drilling sites and oil release characteristics, (3) selection of the simulation periods and collection of environmental data, (4) identification of the species of interest and their characteristics, (5) performance of stochastic calculations and oil contamination probability analysis, (6) determination of the worst-cases, (7) determination of the characteristics of response systems, (8) performance of deterministic calculations, and (9) assessment of the impact of oil spill in the areas of interest. Stochastic calculations that were performed for three typical seasonal weather variations of the year 2015, three oil release sites and specific oil characteristics, showed that there is a considerable probability of oil pollution that reaches 30% in the Mesolongi - Aitoliko lagoons. Based on a simplified approach regarding the characteristic of the sensitive birds and fish in the lagoons, deterministic calculations showed that 78-90% of the bird population and 2-4% of the fish population are expected to be contaminated in the case of an oil spill without any intervention. The use of dispersants reduced the amount of stranded oil by approximately 16-21% and the contaminated bird population of the lagoons to approximately 70%; however, the affected fish population increased to 6-8.5% due to the higher oil concentration in the water column. Mechanical recovery with skimmers "cleaned" almost 10% of the released oil quantity, but it did not have any noticeable effect on the stranded oil and the impacted bird and fish populations.