Operationalising uncertainty in data and models for integrated water resources management.

Key sources of uncertainty of importance for water resources management are (1) uncertainty in data; (2) uncertainty related to hydrological models (parameter values, model technique, model structure); and (3) uncertainty related to the context and the framing of the decision-making process. The European funded project 'Harmonised techniques and representative river basin data for assessment and use of uncertainty information in integrated water management (HarmoniRiB)' has resulted in a range of tools and methods to assess such uncertainties, focusing on items (1) and (2). The project also engaged in a number of discussions surrounding uncertainty and risk assessment in support of decision-making in water management. Based on the project's results and experiences, and on the subsequent discussions a number of conclusions can be drawn on the future needs for successful adoption of uncertainty analysis in decision support. These conclusions range from additional scientific research on specific uncertainties, dedicated guidelines for operational use to capacity building at all levels. The purpose of this paper is to elaborate on these conclusions and anchoring them in the broad objective of making uncertainty and risk assessment an essential and natural part in future decision-making processes.

Uncertainty analysis at large scales: limitations and subjectivity of current practices--a water quality case study.

Uncertainty analysis for large-scale model studies is a challenging activity that requires a different approach to uncertainty analysis at a smaller scale. However, in river basin studies, the practice of uncertainty analysis at a large scale is mostly derived from practice at a small scale. The limitations and inherent subjectivity of some current practices and assumptions are identified, based on the results of a quantitative uncertainty analysis exploring the effects of input data and parameter uncertainty on surface water nutrient concentration. We show that: (i) although the results from small- scale sensitivity analysis are often applied at larger scales, this is not always valid; (ii) the current restriction of the uncertainty assessment to uncertainty types with a strong evidence base gives structurally conservative estimates; (iii) uncertainty due to bias is usually not assessed, but it may easily outweigh the effects of variability; (iv) the uncertainty bandwidth may increase for higher aggregation levels, although the opposite is the standard assumption.

Intramers as promising new tools in functional proteomics.

Aptamers are valuable tools for studying numerous aspects of biological processes, opening up new experimental opportunities to analyse the function of a wide range of cellular molecules. Functional RNA molecules can be rapidly selected in vitro from complex combinatorial mixtures of different sequences. Recently, it was shown that in vitro selection processes can be automated: the first generation selection robots will soon mean aptamers for several targets can be isolated in parallel within days rather than weeks. Aptamers not only exhibit highly specific molecular recognition properties but are also able to modulate the function of their cognate targets in a highly specific manner by agonistic or antagonistic mechanisms. These properties prompted the development of novel technologies to exploit the use of aptamers to modulate distinct functions of biological targets. Recent controlled expression of aptamers inside cells demonstrated their impressive potential as rapidly generated intracellular inhibitors of biomolecules. Intracellularly applied aptamers are also called 'intramers'. Here we discuss recent developments and strategies for intramer-based technologies that have the potential to greatly facilitate characterisation of unknown protein functions in the context of their natural expression status in vivo. Thus, intramer-based technologies offer many promising applications in functional genomics, proteomics and drug discovery.

Aptamers that bind to the antibiotic moenomycin A.

Nuclease-resistant moenomycin-binding aptamers with dissociation constants in the range of 300 to 400 nM have been selected. Competition experiments have demonstrated that these aptamers recognize a disaccharide analogue of moenomycin. The results offer the opportunity of setting up a selective and sensitive assay for identifying moenomycin biosynthetic precursors.

Controlling small guanine-nucleotide-exchange factor function through cytoplasmic RNA intramers.

ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have been implicated in the control of diverse biological functions. Two main classes of positive regulatory elements for ARF have been discovered so far: the large Sec7/Gea and the small cytohesin/ARNO families, respectively. These proteins harbor guanine-nucleotide-exchange factor (GEF) activity exerted by the common Sec7 domain. The availability of a specific inhibitor, the fungal metabolite brefeldin A, has enabled documentation of the involvement of the large GEFs in vesicle transport. However, because of the lack of such tools, the biological roles of the small GEFs have remained controversial. Here, we have selected a series of RNA aptamers that specifically recognize the Sec7 domain of cytohesin 1. Some aptamers inhibit guanine-nucleotide exchange on ARF1, thereby preventing ARF activation in vitro. Among them, aptamer M69 exhibited unexpected specificity for the small GEFs, because it does not interact with or inhibit the GEF activity of the related Gea2-Sec7 domain, a member of the class of large GEFs. The inhibitory effect demonstrated in vitro clearly is observed as well in vivo, based on the finding that M69 produces similar results as a dominant-negative, GEF-deficient mutant of cytohesin 1: when expressed in the cytoplasm of T-cells, M69 reduces stimulated adhesion to intercellular adhesion molecule-1 and results in a dramatic reorganization of F-actin distribution. These highly specific cellular effects suggest that the ARF-GEF activity of cytohesin 1 plays an important role in cytoskeletal remodeling events of lymphoid cells.

Nucleic acid aptamers-from selection in vitro to applications in vivo.

Aptamers are nucleic acid ligands which are isolated from combinatorial oligonucleotide libraries by in vitro selection. They exhibit highly complex and sophisticated molecular recognition properties and are capable of binding tightly and specifically to targets ranging from small molecules to complex multimeric structures. Besides their promising application as molecular sensors, many aptamers targeted against proteins are also able to interfere with the proteins' biological function. Recently developed techniques facilitate the intracellular application of aptamers and their use as in vivo modulators of cellular physiology. Using these approaches, one can quickly obtain highly specific research reagents that act on defined intracellular targets in the context of the living cell.

Cytoplasmic RNA modulators of an inside-out signal-transduction cascade.

A vaccinia virus-based RNA expression system enabled high-level cytoplasmic expression of RNA aptamers directed against the intracellular domain of the beta2 integrin LFA-1, a transmembrane protein that mediates cell adhesion to intercellular adhesion molecule-1 (ICAM-1). In two different cell types, cytoplasmic expression of integrin-binding aptamers reduced inducible cell adhesion to ICAM-1. The aptamers specifically target, and thereby define, a functional cytoplasmic subdomain important for the regulation of cell adhesion in leukocytes. Our approach of aptamer-controlled blocking of signaling pathways in vivo could potentially be applied wherever targeted modulation of a signal-transduction cascade is desired.