A functional gene array for detection of bacterial virulence elements

Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples.     

Structure and evolution of the Cinful retrotransposon family of maize.

A maize cDNA clone was isolated by virtue of its intense hybridization to total maize genomic DNA, indicating homology to highly repetitive sequences. Genomic homologues were identified and subcloned from an adh1-bearing maize yeast artificial chromosome (YAC). Sequencing revealed that the expressed sequence was part of a Ty3-gypsy-type retrotransposon. We discovered and sequenced two complete retrotransposons of this family, and named them Cinful elements because they are members of a family of maize retrotransposons including Zeon-1 and the first plant transposable element sequenced, the solo long terminal repeat (LTR) called Cin1. All are defective, as Cinful-1 and Cinful-2 elements lack gag and Zeon-1 lacks pol homology. Despite the apparent lack of an intact "autonomous" element, the Cinful family has expanded to a copy number of about 18 000, representing just under 9% of the maize genome. Both point mutations and major rearrangements, including possible gene acquisition, differentiate members of the Cinful family. Cinful family members were found to have an unusual feature that we also observed in two other Ty3-class retrotransposons of teosinte and tobacco: related tandem repeats that separate their internal domains with a gag- or pol-containing homology from a 3' segment of unknown function. The conserved and variable features identified provide insights into the origin, mutational history, and functional components of this major constituent of the maize genome.     

Asymmetry in the assembly of the RNAi enzyme complex

A key step in RNA interference (RNAi) is assembly of the RISC, the protein-siRNA complex that mediates target RNA cleavage. Here, we show that the two strands of an siRNA duplex are not equally eligible for assembly into RISC. Rather, both the absolute and relative stabilities of the base pairs at the 5' ends of the two siRNA strands determine the degree to which each strand participates in the RNAi pathway. siRNA duplexes can be functionally asymmetric, with only one of the two strands able to trigger RNAi. Asymmetry is the hallmark of a related class of small, single-stranded, noncoding RNAs, microRNAs (miRNAs). We suggest that single-stranded miRNAs are initially generated as siRNA-like duplexes whose structures predestine one strand to enter the RISC and the other strand to be destroyed. Thus, the common step of RISC assembly is an unexpected source of asymmetry for both siRNA function and miRNA biogenesis.     

Semantic Similarity in Biomedical Ontologies

In recent years, ontologies have become a mainstream topic in biomedical research. When biological entities are described using a common schema, such as an ontology, they can be compared by means of their annotations. This type of comparison is called semantic similarity, since it assesses the degree of relatedness between two entities by the similarity in meaning of their annotations. The application of semantic similarity to biomedical ontologies is recent; nevertheless, several studies have been published in the last few years describing and evaluating diverse approaches. Semantic similarity has become a valuable tool for validating the results drawn from biomedical studies such as gene clustering, gene expression data analysis, prediction and validation of molecular interactions, and disease gene prioritization.     

Developmentally regulated sphingolipid synthesis in African trypanosomes

Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase. Procyclic trypanosomes contain IPC, but also sphingomyelin, while surprisingly bloodstream-stage parasites contain sphingomyelin and ethanolamine phosphorylceramide (EPC), but no detectable IPC. In vivo fluorescent ceramide labelling confirmed stage-specific biosynthesis of both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of TbSLS1-4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels were increased more than threefold by silencing suggesting a toxic downstream effect mediated by this potent intracellular messenger. Topology predictions support a revised six-transmembrane domain model for the kinetoplastid sphingolipid synthases consistent with the proposed mammalian sphingomyelin synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites.     

Differential effect of three-repeat and four-repeat tau on mitochondrial axonal transport

Tau protein is present in six different splice forms in the human brain and interacts with microtubules via either 3 or 4 microtubule binding repeats. An increased ratio of 3 repeat to 4 repeat isoforms is associated with neurodegeneration in inherited forms of frontotemporal dementia. Tau over-expression diminishes axonal transport in several systems, but differential effects of 3 repeat and 4 repeat isoforms have not been studied. We examined the effects of tau on mitochondrial transport and found that both 3 repeat and 4 repeat tau change normal mitochondrial distribution within the cell body and reduce mitochondrial localization to axons; 4 repeat tau has a greater effect than 3 repeat tau. Further, we observed that the 3 repeat and 4 repeat tau cause different alterations in retrograde and anterograde transport dynamics with 3 repeat tau having a slightly stronger effect on axon transport dynamics. Our results indicate that tau-induced changes in axonal transport may be an underlying theme in neurodegenerative diseases associated with isoform specific changes in tau's interaction with microtubules.     

A global analysis of the determinants of alien geographical range size in birds

Aim Determining the causes of range size variation in the distributions of alien species is important for understanding the spread of invasive species. Factors influencing alien range size have been explored for some species at a regional level, but to date there has been no global analysis of an entire class. Here, we present such an analysis for birds, testing for the effects of introduction event, location and species‐level variables on alien range sizes. Location Global. Methods We used a novel dataset on the global distributions of alien bird species to test for relationships between alien range size and colonization pressure, residence time, extent of the global climatic niche, native range size, body mass and specialization, using a statistical approach based on phylogenetic generalized least squares models. We performed this analysis globally, and for separate biogeographical realms. Results Approximately half of the variation in alien bird range size is explained by colonization pressure in univariate analysis. We identified consistent effects of higher colonization pressure at global and realm levels, as well as support for effects of native range size and residence time. We found less support for effects of body mass, specialization or extent of the global climatic niche on alien range size. Main conclusions Alien bird range sizes are generally small relative to their native range sizes, and many are continuing to expand. Nevertheless, current variation is predictable, most strongly by the event‐level factor of colonization pressure. Whether a species is widespread is a better predictor of alien range size than whether a species could be widespread (estimated by global climatic niche extent), while we also find effects of residence time on alien range size. These relationships may help to identify those alien species that are more likely to spread and hence have greater environmental and economic impacts where they have been introduced.     

Sequence Heterogeneity in Closed Simian Virus 40 Deoxyribonucleic Acid

The heteroduplex molecules formed by self-annealing of denatured, singly nicked simian virus 40 (SV40) deoxyribonucleic acid (DNA) prepared from closed viral DNA were examined by formamide-protein film electron microscopy to test the DNA for sequence homogeneity. Sequence inhomogeneity appears in the heteroduplexes as single-strand loops. These result from sequence deletion or from sequence substitution, if regions greater than 50 nucleotides are involved. The undenatured DNA from viruses passaged twice at multiplicities of infection much less than 1 plaque-forming unit (PFU) per cell appeared to be homogeneous in size. The heteroduplexes formed by this DNA indicated that approximately 2% of the molecules carried deletions, but that substitutions were below the level of detection. In contrast, undenatured DNA from viruses grown by passaging undiluted lysates seven times or by infection with stock virus at a multiplicity of infection of 5 PFU per cell contained a large frequency of molecules shorter than the full length. The heteroduplex samples indicated that 12 and 7% of the undenatured material contained base substitutions, and 13 and 11% contained deletions. The deletions and substitutions appear to occur in separate molecules. Length measurements on heteroduplexes displaying the loop characteristic of substitutions have established that these molecules are from true sequence substitutions, and not from adjacent or overlapping deletions. More than 80% of the molecules carrying substitutions are shorter than the native SV40 length. On the average, the substituted sequence is about 20% of the length of SV40, but it replaces a sequence about 30% of the native length. The substituted sequences may be host cell nuclear DNA, possibly arising from integration of SV40 into the chromosome followed by excision of the SV40 DNA together with chromosomal DNA.     

Production of recombinant serpins in Escherichia coli

Expression systems based on Escherichia coli offer fast, cheap, and convenient means for the production of recombinant serpins. Over 30 active serpins from prokaryotic and eukaryotic organisms have been produced in this way, using a variety of vectors, promoters, fusion partners, and host strains. Serpins forming insoluble inclusion bodies in E. coli can generally be solubilized and refolded. Here, we outline the general approaches and procedures to be considered when contemplating the use of E. coli for recombinant serpin production.