EASI—enrichment of alternatively spliced isoforms

Alternative splicing produces more than one protein from the majority of genes and the rarer forms can have dominant functions. Instability of alternative transcripts can also hinder the study of regulation of gene expression by alternative splicing. To investigate the true extent of alternative splicing we have developed a simple method of enriching alternatively spliced isoforms (EASI) from PCRs using beads charged with Thermus aquaticus single-stranded DNA-binding protein (T.Aq ssb). This directly purifies the single-stranded regions of heteroduplexes between alternative splices formed in the PCR, enabling direct sequencing of all the rare alternative splice forms of any gene. As a proof of principle the alternative transcripts of three tumour suppressor genes, TP53, MLH1 and MSH2, were isolated from testis cDNA. These contain missing exons, cryptic splice sites or include completely novel exons. EASI beads are stable for months in the fridge and can be easily combined with standard protocols to speed the cloning of novel transcripts

Interception and Offshore Processing of Asylum Seekers: The International Law Dimensions

For decades the international community has conducted a delicate and politically charged balancing act trying to reconcile the inexorable increase in refugees-and the need to find permanent homes for them with the fundamental right of all countries to have secure frontiers. While the notion of non-refoulement remains fundamental to the treatment of asylum seekers, their rights vis A vis the states in which they seek asylum are significantly circumscribed by their alien status. States have a right to control entry to their territories. In the development of asylum law and policy, the central difficulty for states, and indeed the international community, is how to construct an appropriate balance between the urgent humanitarian demands to protect those who are genuinely in need of asylum, and the exclusion of those who do not qualify for humanitarian protection

Development of Vegetation Communities in a Retrogressive Thaw Slump near Mayo, Yukon Territory : A 10-year Assessment

The vegetation in a retrogressive thaw slump, first surveyed and documented in 1987, was revisited 10 years later to investigate its subsequent development and to test a chronosequence-based successional model. The thaw slump stabilized in 1994, when the headwall became covered by organic and mineral debris. As a result, the meltwater supply from headwall ablation ceased. Alteration of environmental conditions due to stabilization of the headwall diverted the vegetation succession from the chronosequence determined in 1987. Areas that were marshy in 1987 dried up, and an area dominated by Polygonum alaskanum appeared close to the headwall. Much of the thaw slump was dominated by Salix spp. in 1997, rather than the Equisteum of 1987. However, the ground more than 200 m from the headwall, over a decade old in 1987, experienced less change in edaphic conditions, and the communities there continued to develop a structure approaching the surrounding undisturbed forest.La végétation située dans un décrochement de fonte régressif, étudiée et documentée pour la première fois en 1987, a fait l'objet d'une autre étude sur place dix ans plus tard pour examiner son évolution et tester un modèle de succession fondé sur la chronoséquence. Le décrochement de fonte s'est stabilisé en 1994, quand le mur de rimaye s'est couvert de débris organiques et minéraux. Il en a résulté que l'alimentation en eau de fonte venant de l'ablation du mur de rimaye a cessé. Le changement dans les conditions environnementales dû à la stabilisation du mur a détourné la succession végétale de la chronoséquence établie en 1987. Des zones qui étaient marécageuses en 1987 se sont asséchées, et une étendue où dominait Polygonum alaskanum apparue près du mur. En 1997, une grande partie de la zone du décrochement de fonte était dominée par des espèces Salix, plutôt qu'Equisteum, comme c'était le cas en 1987. Toutefois, le sol situé à plus de 200 m du mur de rimaye, sol vieux de dix ans en 1987, a subi moins de changement dans les conditions édaphiques, et les communautés y ont continué à évoluer vers une structure qui se rapproche de la forêt non perturbée située à la périphérie

In Vitro Production of Ergothioneine Isotopologues

Ergothioneine is an emerging component of the redox homeostasis system in human cells and in microbial pathogens, such as Mycobacterium tuberculosis and Burkholderia pseudomallei. The synthesis of stable isotope-labeled ergothioneine derivatives may provide important tools for deciphering the distribution, function, and metabolism of this compound in vivo. We describe a general protocol for the production of ergothioneine isotopologues with programmable 2 H, 15 N, 13 C, 34 S, and 33 S isotope labeling patterns. This enzyme-based approach makes efficient use of commercial isotope reagents and is also directly applicable to the synthesis of radio-isotopologues

An inverse modeling approach to obtain P-T conditions of metamorphic stages involving garnet growth and resorption

This contribution presents an approach and a computer program (GRTMOD) for numerical simulation of garnet evolution based on compositions of successive growth zones in natural samples. For each garnet growth stage, a new local effective bulk composition is optimized, allowing for resorption and/or fractionation of previously crystallized garnet. The successive minimizations are performed using the Nelder-Mead algorithm; a heuristic search method. An automated strategy including two optimization stages and one refinement stage is described and tested. This program is used to calculate pressure-temperature (P-T) conditions of crystal growth as archived in garnet from the Sesia Zone (Western Alps). The compositional variability of successive growth zones is characterized using standardized X-ray maps and the program XMapTools. The model suggests that Permian garnet cores crystallized under granulite-facies conditions at T>800 °C and P = 6 kbar. During Alpine times, a first garnet rim grew at eclogite-facies conditions (650 °C, 16 kbar) at the expense of the garnet core. A second rim was added at lower P (∼11 kbar) and 630 °C. In total, garnet resorption is modeled to amount to ∼9 vol% during the Alpine evolution; this value is supported by our observations in X-ray compositional maps

Structural and Mechanistic Basis for Anaerobic Ergothioneine Biosynthesis

Ergothioneine is an emergent factor in cellular redox biochemistry in humans and pathogenic bacteria. Broad consensus has formed around the idea that ergothioneine protects cells against reactive oxygen species. The recent discovery that anaerobic microorganisms make the same metabolite using oxygen-independent chemistry indicates that ergothioneine also plays physiological roles under anoxic conditions. In this report, we describe the crystal structure of the anaerobic ergothioneine biosynthetic enzyme EanB from green sulfur bacterium Chlorobium limicola. This enzyme catalyzes the oxidative sulfurization of N-alpha-trimethyl histidine. On the basis of structural and kinetic evidence, we describe the catalytic mechanism of this unusual C-S bond-forming reaction. Significant active-site conservation among distant EanB homologues suggests that the oxidative sulfurization of heterocyclic substrates may occur in a broad range of bacteria