Revised Selection Criteria for Candidate Restriction Enzymes in Genome Walking

A new method to improve the efficiency of flanking sequence identification by genome walking was developed based on an expanded, sequential list of criteria for selecting candidate enzymes, plus several other optimization steps. These criteria include: step (1) initially choosing the most appropriate restriction enzyme according to the average fragment size produced by each enzyme determined using in silico digestion of genomic DNA, step (2) evaluating the in silico frequency of fragment size distribution between individual chromosomes, step (3) selecting those enzymes that generate fragments with the majority between 100 bp and 3,000 bp, step (4) weighing the advantages and disadvantages of blunt-end sites vs. cohesive-end sites, step (5) elimination of methylation sensitive enzymes with methylation-insensitive isoschizomers, and step (6) elimination of enzymes with recognition sites within the binary vector sequence (T-DNA and plasmid backbone). Step (7) includes the selection of a second restriction enzyme with highest number of recognition sites within regions not covered by the first restriction enzyme. Step (8) considers primer and adapter sequence optimization, selecting the best adapter-primer pairs according to their hairpin/dimers and secondary structure. In step (9), the efficiency of genomic library development was improved by column-filtration of digested DNA to remove restriction enzyme and phosphatase enzyme, and most important, to remove small genomic fragments (<100 bp) lacking the T-DNA insertion, hence improving the chance of ligation between adapters and fragments harbouring a T-DNA. Two enzymes, NsiI and NdeI, fit these criteria for the Arabidopsis thaliana genome. Their efficiency was assessed using 54 T3 lines from an Arabidopsis SK enhancer population. Over 70% success rate was achieved in amplifying the flanking sequences of these lines. This strategy was also tested with Brachypodium distachyon to demonstrate its applicability to other larger genomes

Assessing the state of marine biodiversity in the Northeast Atlantic

The Northeast Atlantic, a highly productive maritime area, has been exposed to a wide range of direct human pressures, such as fishing, shipping, coastal development, pollution, and non-indigenous species (NIS) introductions, in addition to anthropogenically-driven global climate change. Nonetheless, this regional sea supports a high diversity of species and habitats, whose functioning provides a variety of ecosystem services, essential for human welfare. In 2017, OSPAR, the Northeast Atlantic Regional Seas Commission, delivered an assessment of marine biodiversity for the Northeast Atlantic. This assessment examined biodiversity indicators separately to identify changes in Northeast Atlantic biodiversity, but stopped short of determining the status of biodiversity for many species and habitats. Here, we expand on this work and for the first time, a semi-quantitative approach is applied to evaluate holistically the state of Northeast Atlantic marine biodiversity across marine food webs, from plankton to top predators, via fish, pelagic and benthic habitats, including xeno-biodiversity (i.e. NIS). Our analysis reveals widespread degradation in marine ecosystems and biodiversity, particularly for marine birds and coastal bottlenose dolphins, as well as for benthic habitats and fish in some regions. The poor biodiversity status of these ecosystem components is likely the result of cumulative effects of human activities, such as habitat destruction or disturbance, overexploitation, eutrophication, the introduction of NIS, and climate change. Bright spots are also revealed, such as recent signs of recovery in some fish and marine bird communities and recovery in harbour and grey seal populations and the condition of coastal benthic communities in some regions. The status of many indicators across all ecosystem components, but particularly for the novel pelagic habitats, food webs and NIS indicators, however, remains uncertain due to gaps in data, unclear pressure-state relationships, and the non-linear influence of some pressures on biodiversity indicators. Improving monitoring and data access and increasing understanding of pressure-state relationships, including those that are non-linear, is therefore a priority for enabling future assessments, as is consistent and stable resourcing for expert involvement

Proposed approaches for indicator integration. EcApRHA Deliverable WP 4.1

Executive SummaryThe Marine Strategy Framework Directive (MSFD) aims to achieve Good Environmental Status (GES) withinEuropean Commission waters through an ecosystem‐based approach. The MSFD requires Member Statessharing a marine region or sub‐region to cooperate to ensure that the Directive’s objectives are achievedWorking towards an ecosystem perspective: Proposals for the integration of pelagic, benthic and food web indicatorsand to coordinate their actions through Regional Seas Conventions e.g. the OSPAR Commission for theNorth‐East Atlantic. As part of the ‘applying an Ecosystem Approach to (sub) Regional Habitat Assessments’(EcApRHA) project, integration of indicators under Descriptor 1 (biodiversity), 4 (food webs) and 6 (seafloorintegrity), relating to pelagic and benthic habitats and food webs have been forwarded to work towards anecosystem’s approach in assessing habitats regionally. The content of this report covers differentapproaches developed to integrate indicators forwarded within the project.Five methods are described, four of which were developed to integrate indicators developed under theEcApRHA project. The fifth, OSPAR’s cumulative effect approach has also been summarised as an additionalapproach to integrate indicators. For each method, management implications; the advantages anddisadvantages in relation to being able to work toward assessment of GES; and the confidence in theassessments, are highlighted. The time it would take for the approach to become fully operational, itsfeasibility and costs are also discussed.From the five methods described, three main approaches are discussed:I. A quantitative method to draw links between indicators to assess pressures that have effectson the different aspects of the marine ecosystem (Chapters 3‐4).II. Use of the Nested Environmental status Assessment Tool (NEAT) to integrate differentindicators to provide an overall assessment (Chapters 5 and 6).III. Use of an industry led risk assessment tool (Bow‐Ties) to assess cumulative effects (Chapter 7).The integration approaches outlined within this document demonstrate the developments made within theEcApRHA project to ensure the various indicators under the different descriptors are not only operational,but also integrated in a way which permits a more holistic assessment of the marine environment. Usingsuch a two‐tiered approach of individual indicator and integrated analysis, will enable an understanding ofwhy certain aspects of the marine environment may not be in good condition, and thereby recommendspecific management measures to ameliorate them. Although the approaches forwarded have been initiallytrialled in the North‐East Atlantic, they are able to be applied to other MSFD Regional seas areas. Eachmethod addresses different levels of integration (indicator, habitat or ecosystem) and requires furtherdevelopment and testing. They should be thus considered as complementary and gaps should beprogressed in parallel to ensure coherent progress towards an overall ecosystem approach. In addition,with some further comparative testing between the different methods outlined within this document,options to continue forwarding integrated assessment of OSPAR indicators could be proposed. Themethods outlined within this document are a first step in applying an ecosystem’s approach to assessingthe state of our seas

Long-term Transgene Expression in the Central Nervous System Using DNA Nanoparticles

This study demonstrates proof of concept for delivery and expression of compacted plasmid DNA in the central nervous system. Plasmid DNA was compacted with polyethylene glycol substituted lysine 30-mer peptides, forming rod-like nanoparticles with diameters between 8 and 11 nm. Here we show that an intracerebral injection of compacted DNA can transfect both neurons and glia, and can produce transgene expression in the striatum for up to 8 weeks, which was at least 100-fold greater than intracerebral injections of naked DNA plasmids. Bioluminescent imaging (BLI) of injected animals at the 11th postinjection week revealed significantly higher transgene activity in animals receiving compacted DNA plasmids when compared to animals receiving naked DNA. There was minimal evidence of brain inflammation. Intrastriatal injections of a compacted plasmid encoding for glial cell line–derived neurotrophic factor (pGDNF) resulted in a significant overexpression of GDNF protein in the striatum 1–3 weeks after injection