Characterization of the 2'-5'-oligoadenylate synthetase ubiquitin-like family

The interferon-induced 2′–5′-oligoadenylate synthetases (OAS) are important for the antiviral activity of interferons. The human and murine OAS gene families each contain four genes: OAS1, OAS2, OAS3 and OASL, all having one or more conserved OAS units composed of five translated exons. The OASL gene has both an OAS unit and a C-terminus of two ubiquitin-like repeats. In this study, we demonstrate that murine Oasl1 protein is inactive while murine Oasl2 is active as an OAS. Further more, murine Oasl2 requires double-stranded RNA as co-factor. The affinity of murine Oasl2 for the double-stranded RNA activator is higher than that of human OAS1 (p42 isoform). We propose a model for the evolutionary origin of the murine Oasl1 and Oasl2 genes. The identification of a human orthologue (hOASL2) to the murine Oasl2 gene establishes that the OASL gene was duplicated prior to the radiation of the rodent and primate groups. We suggest that murine Oasl2, which has both enzymatic activity and a ubiquitin-like domain, is a functional intermediate between the active OAS species and the inactive human OASL1/murine Oasl1 proteins. In addition, we propose that murine Oasl1 appears to have gained a hitherto uncharacterized function independent of 2′–5′-linked oligoadenylate synthesis.     

Suppression of eukaryotic translation termination by selected RNAs

Using selection-amplification, we have isolated RNAs with affinity for translation termination factors eRF1 and eRF1.eRF3 complex. Individual RNAs not only bind, but inhibit eRF1-mediated release of a model nascent chain from eukaryotic ribosomes. There is also significant but weaker inhibition of eRF1-stimulated eRF3 GTPase and eRF3 stimulation of eRF1 release activity. These latter selected RNAs therefore hinder eRF1.eRF3 interactions. Finally, four RNA inhibitors of release suppress a UAG stop codon in mammalian extracts dependent for termination on eRF1 from several metazoan species. These RNAs are therefore new specific inhibitors for the analysis of eukaryotic termination, and potentially a new class of omnipotent termination suppressors with possible therapeutic significance.     

Recovery and virulence phenotyping of the historic ‘Stubbs collection’ of the yellow rust fungus Puccinia striiformis from wheat

A unique collection of spore samples of Puccinia striiformis, often referred to as the ‘Stubbs collection’, has been stored in liquid nitrogen from 18 to 45 years. A subset of samples representing 35 countries and 28 years was investigated to assess recovery rate, race identity and previously undetected virulence. A new method for recovery using an airbrush sprayer and Novec? 7100 for inoculating the host plants was highly successful. Ninety‐six percent of 231 isolates were recovered. Virulence phenotyping was done using differential sets of wheat genotypes representing specific‐resistance genes. A total of 181 samples represented single genotypes (isolates), whereas 40 samples consisted of at least two genotypes. Race identity was confirmed for 102 of 181 single‐genotype isolates. The virulence phenotype was updated for additional 44 isolates based on improved resolution of results because of updated and more informative wheat‐differential sets. The remaining 35 isolates showed discrepancies for one or more virulences when compared with past results. Additional virulences corresponding to Yr17, Yr25 and Yr27, respectively, which were not assayed originally, were discovered. The value of biological collections for research and plant breeding is discussed along with the challenges of maintaining collections of biotrophic microorganisms.     

Optimization and clinical validation of a pathogen detection microarray

DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Origin,Migration Routes and Worldwide Population Genetic Structure of the Wheat Yellow Rust Pathogen Puccinia striiformis f.sp. tritici

Analyses of large-scale population structure of pathogens enable the identification of migration patterns, diversity reservoirs or longevity of populations, the understanding of current evolutionary trajectories and the anticipation of future ones. This is particularly important for long-distance migrating fungal pathogens such as Puccinia striiformis f.sp. tritici (PST), capable of rapid spread to new regions and crop varieties. Although a range of recent PST invasions at continental scales are well documented, the worldwide population structure and the center of origin of the pathogen were still unknown. In this study, we used multilocus microsatellite genotyping to infer worldwide population structure of PST and the origin of new invasions based on 409 isolates representative of distribution of the fungus on six continents. Bayesian and multivariate clustering methods partitioned the set of multilocus genotypes into six distinct genetic groups associated with their geographical origin. Analyses of linkage disequilibrium and genotypic diversity indicated a strong regional heterogeneity in levels of recombination, with clear signatures of recombination in the Himalayan (Nepal and Pakistan) and near-Himalayan regions (China) and a predominant clonal population structure in other regions. The higher genotypic diversity, recombinant population structure and high sexual reproduction ability in the Himalayan and neighboring regions suggests this area as the putative center of origin of PST. We used clustering methods and approximate Bayesian computation (ABC) to compare different competing scenarios describing ancestral relationship among ancestral populations and more recently founded populations. Our analyses confirmed the Middle East-East Africa as the most likely source of newly spreading, high-temperature-adapted strains; Europe as the source of South American, North American and Australian populations; and Mediterranean-Central Asian populations as the origin of South African populations. Although most geographic populations are not markedly affected by recent dispersal events, this study emphasizes the influence of human activities on recent long-distance spread of the pathogen.     

Biomechanical spinal growth modulation and progressive adolescent scoliosis – a test of the 'vicious cycle' pathogenetic hypothesis: Summary of an electronic focus group debate of the IBSE

There is no generally accepted scientific theory for the causes of adolescent idiopathic scoliosis (AIS). As part of its mission to widen understanding of scoliosis etiology, the International Federated Body on Scoliosis Etiology (IBSE) introduced the electronic focus group (EFG) as a means of increasing debate on knowledge of important topics. This has been designated as an on-line Delphi discussion. The text for this debate was written by Dr Ian A Stokes. It evaluates the hypothesis that in progressive scoliosis vertebral body wedging during adolescent growth results from asymmetric muscular loading in a "vicious cycle" (vicious cycle hypothesis of pathogenesis) by affecting vertebral body growth plates (endplate physes). A frontal plane mathematical simulation tested whether the calculated loading asymmetry created by muscles in a scoliotic spine could explain the observed rate of scoliosis increase by measuring the vertebral growth modulation by altered compression. The model deals only with vertebral (not disc) wedging. It assumes that a pre-existing scoliosis curve initiates the mechanically-modulated alteration of vertebral body growth that in turn causes worsening of the scoliosis, while everything else is anatomically and physiologically 'normal' The results provide quantitative data consistent with the vicious cycle hypothesis. Dr Stokes' biomechanical research engenders controversy. A new speculative concept is proposed of vertebral symphyseal dysplasia with implications for Dr Stokes' research and the etiology of AIS. What is not controversial is the need to test this hypothesis using additional factors in his current model and in three-dimensional quantitative models that incorporate intervertebral discs and simulate thoracic as well as lumbar scoliosis. The growth modulation process in the vertebral body can be viewed as one type of the biologic phenomenon of mechanotransduction. In certain connective tissues this involves the effects of mechanical strain on chondrocytic metabolism a possible target for novel therapeutic intervention.