The importance of host plant-habitat substrate in the maintenance of a unique isolate of the Sandhill Rustic: disturbance, shingle matrix and bare ground indicators

The Sandhill Rustic moth Luperina nickerlii leechi is an isolate restricted to the beach of Loe Bar in Cornwall UK; it is a UK Priority taxon because of its unique status and small, declining population. The larval foodplant is Sand Couch-grass Elytrigia juncea; the larvae feed at first inside the stems and then on the rhizomes underground. The distribution of the moth and E. juncea were investigated in this study in relation to substrate conditions and other plants. E. juncea has an above-ground clumped distribution; near the sea the plants are linked by rhizomes, but away from the sea the apparent clumping is a probable historic record of previous connectivity where the rhizomes have disappeared. The adult moths were found to be associated with more vigorous E. juncea plants growing in sparsely vegetated, more disturbed ground near the sea, especially those plants with large rhizome systems along which later instar larvae can travel between plants. Emerging moths were associated with areas of extensive E. juncea surrounded by bare ground, where there was a substantial variation of shingle particles between 125?μm and 8?mm. The absence of vegetation and associated roots apart from E. juncea, and a coarse particle mix, may allow larvae to move through the shingle and the moths to push their way upwards from their subterranean pupae. We conclude that management should aim to create areas of bare ground with extensive patches of E. juncea growing in coarser shingle, with extensive underground connectivity and few other plants.     

On temporal partitioning of a community of ectomycorrhizal fungi

Several mechanisms may contribute to the high species richness often reported in ectomycorrhizal (ECM) fungal communities, including spatial and temporal partitioning. Here, we focus on temporal partitioning. Using molecular methods, we determined the frequencies of occurrence of ECM fungal species detected as hyphae and ECM roots in the forest floor of a Pinus resinosa plantation during a 13-month period. We then used a novel statistical procedure to place the most frequently occurring ECM fungal species into groups distinguished by their patterns of relative frequency over time. Three groups with contrasting temporal patterns were distinguishable for fungal species detected as hyphae. Two groups were distinguishable for species detected as ECM roots. Our results support the hypothesis that temporal partitioning occurs among the species of ECM fungi in this community, but we did not address its causes, which may have involved interactions among species' physiological tolerances, temporal environmental variability, temporal patterns of root production, and variation in fungal genet lifespan. These interactions should be the subjects of future research.     

A research agenda for helminth diseases of humans: diagnostics for control and elimination programmes

Diagnostic tools appropriate for undertaking interventions to control helminth infections are key to their success. Many diagnostic tests for helminth infection have unsatisfactory performance characteristics and are not well suited for use in the parasite control programmes that are being increasingly implemented. Although the application of modern laboratory research techniques to improve diagnostics for helminth infection has resulted in some technical advances, uptake has not been uniform. Frequently, pilot or proof of concept studies of promising diagnostic technologies have not been followed by much needed product development, and in many settings diagnosis continues to rely on insensitive and unsatisfactory parasitological or serodiagnostic techniques. In contrast, PCR-based xenomonitoring of arthropod vectors, and use of parasite recombinant proteins as reagents for serodiagnostic tests, have resulted in critical advances in the control of specific helminth parasites. The Disease Reference Group on Helminths Infections (DRG4), established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR) was given the mandate to review helminthiases research and identify research priorities and gaps. In this review, the diagnostic technologies relevant to control of helminth infections, either available or in development, are reviewed. Critical gaps are identified and opportunities to improve needed technologies are discussed.     

Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1)

Heparan sulfate/heparin N-deacetylase/N-sulfotransferase-1 (NDST-1) is a critical enzyme involved in heparan sulfate/heparin biosynthesis. This dual-function enzyme modifies the GlcNAc-GlcA disaccharide repeating sugar backbone to make N-sulfated heparosan. N-sulfation is an absolute requirement for the subsequent epimerization and O-sulfation steps in heparan sulfate/heparin biosynthesis. We have expressed rat liver (r) NDST-1 in Saccharomyces cerevisiae as a soluble protein. The yeast-expressed enzyme has both N-deacetylase and N-sulfotransferase activities. N-acetyl heparosan, isolated from Escherichia coli K5 polysaccharide, de-N-sulfated heparin (DNSH) and completely desulfated N-acetylated heparan sulfate (CDSNAcHS) are all good substrates for the rNDST-1. However, N-desulfated, N-acetylated heparin (NDSNAcH) is a poor substrate. The rNDST-1 was partially purified on heparin Sepharose CL-6B. Purified rNDST-1 requires Mn(2+) for its enzymatic activity, can utilize PAPS regenerated in vitro by the PAPS cycle (PAP plus para-nitrophenylsulfate in the presence of arylsulfotransferase IV), and with the addition of exogenous PAPS is capable of producing 60-65% N-sulfated heparosan from E. coli K5 polysaccharide or Pasteurella multocida polysaccharide.     

2,6-disubstituted pyran-4-one and thiopyran-4-one inhibitors of DNA-Dependent protein kinase (DNA-PK)

6-aryl-2-morpholin-4-yl-4H-pyran-4-ones and 6-aryl-2-morpholin-4-yl-4H-thiopyran-4-ones were synthesised and evaluated as potential inhibitors of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). Several compounds in each series exhibited superior activity to the chromenone LY294002, and were of comparable potency to the benzochromenone NU7026 (IC(50)=0.23 microM). Importantly, members of both structural classes were found to be selective inhibitors of DNA-PK over related phosphatidylinositol 3-kinase-related kinase (PIKK) family members. A multiple-parallel synthesis approach, employing Suzuki cross-coupling methodology, was utilised to prepare libraries of thiopyran-4-ones with a range of aromatic groups at the 3'- and 4'-positions on the thiopyran-4-one 6-aryl ring. Screening of the libraries resulted in the identification of 6-aryl-2-morpholin-4-yl-4H-thiopyran-4-ones bearing naphthyl or benzo[b]thienyl substituents at the 4'-position, as potent DNA-PK inhibitors with IC(50) values in the 0.2-0.4 microM range.     

Computational Finishing of Large Sequence Contigs Reveals Interspersed Nested Repeats and Gene Islands in the rf1-Associated Region of Maize

The architecture of grass genomes varies on multiple levels. Large long terminal repeat retrotransposon clusters occupy significant portions of the intergenic regions, and islands of protein-encoding genes are interspersed among the repeat clusters. Hence, advanced assembly techniques are required to obtain completely finished genomes as well as to investigate gene and transposable element distributions. To characterize the organization and distribution of repeat clusters and gene islands across large grass genomes, we present 961- and 594-kb contiguous sequence contigs associated with the rf1 (for restorer of fertility1) locus in the near-centromeric region of maize (Zea mays) chromosome 3. We present two methods for computational finishing of highly repetitive bacterial artificial chromosome clones that have proved successful to close all sequence gaps caused by transposable element insertions. Sixteen repeat clusters were observed, ranging in length from 23 to 155 kb. These repeat clusters are almost exclusively long terminal repeat retrotransposons, of which the paleontology of insertion varies throughout the cluster. Gene islands contain from one to four predicted genes, resulting in a gene density of one gene per 16 kb in gene islands and one gene per 111 kb over the entire sequenced region. The two sequence contigs, when compared with the rice (Oryza sativa) and sorghum (Sorghum bicolor) genomes, retain gene colinearity of 50% and 71%, respectively, and 70% and 100%, respectively, for high-confidence gene models. Collinear genes on single gene islands show that while most expansion of the maize genome has occurred in the repeat clusters, gene islands are not immune and have experienced growth in both intragene and intergene locations.Genome sequencing of the maize (Zea mays) genome is nearing completion (Bennetzen et al., 2001; Chandler and Brendel, 2002; Wessler, 2006); it is the largest and most difficult-to-assemble plant genome sequenced to date. Maize is an important economic, agricultural, industrial, and research crop; however, with a genome close to the size of the human genome (2.8 Gb) and its high percentage of repetitive elements, acquiring the maize genome seemed a daunting task. Approximately 67% of the genome is made up of transposable elements (TEs; Haberer et al., 2005; Kronmiller and Wise, 2008), increasing the difficulty of assembly (Rabinowicz and Bennetzen, 2006). Much exploratory work has gone into isolating and sequencing just the gene areas and ignoring the repetitive regions, both by methylation filtration (Rabinowicz et al., 1999; Palmer et al., 2003; Whitelaw et al., 2003) and high-C₀t (Whitelaw et al., 2003; Yuan et al., 2003) systems, which have assisted researchers with selecting only genic regions to sequence. These methods have captured a majority of the maize genic sequence (Fu et al., 2005), but they still have the potential to miss important regions. The current genome-sequencing project aims to capture the entire gene set of maize, including regulatory regions. However, the current strategy will not provide a fully assembled genome but rather assembled bacterial artificial chromosome (BAC) contigs ordered and orientated to provide complete gene regions that are adjacent to potentially incomplete TE clusters.The landscape of the maize genome provides an interesting challenge for both sequencing and subsequent annotation. A high density of long terminal repeat (LTR) retrotransposons has had a direct effect on the genome size of many plant genomes, including maize (SanMiguel et al., 1996; Bennetzen et al., 2005; Hawkins et al., 2006; Piegu et al., 2006). Besides expanding genome size, LTR retrotransposons can have an impact on evolution of the species (Kidwell and Lisch, 2000). LTR retrotransposon insertions tend to form nested clusters (SanMiguel and Bennetzen, 1998), which are separated by small regions of several genes. Large nested repeat clusters consist of TE insertions inside TE sequences, expanding the repeat cluster and breaking up the sequence of the TEs found within, hindering repeat and gene annotation and increasing the difficulty of assembly. However, full sequence completion of the repetitive regions can be of great benefit to understanding the evolutionary history of the maize genome. LTR retrotransposons can provide an estimated time since insertion by calculating the divergence of their LTRs (Kimura, 1980; Ma and Bennetzen, 2004), and carefully sequenced assemblies of nested repeat clusters can help to illustrate their expansion, proliferation, and evolution across the genome (Kronmiller and Wise, 2008).Previous studies of large contiguous regions of maize have provided a general view of the landscape of the genome. Unfinished sequence totaling 7.8 Mb from chromosome 1 and 6.6 Mb from chromosome 9 shows a gene density of one gene per 33 and 27 kb, respectively (Bruggmann et al., 2006). BAC contigs ranging in size from 126 to 405 kb show a gene density of one gene per 19 kb and genes found in small groups between large repeat clusters (Brunner et al., 2005). Genome-wide analysis of maize BACs has painted a different picture: while gene density of 100 random BACs at one gene per 44 kb was similar to the above results, genes were not observed in tight clusters (Haberer et al., 2005). When investigating gene-specific areas of maize, this dichotomy of gene density is also seen. Analysis of gene-rich regions such as the 22-kd α-zein gene family on maize chromosome 4 reveals a high density of genes, with one gene observed per 10 kb over 346 kb (Song et al., 2001). The Adh1 locus on maize chromosome 1 contains two genes across 280 kb, or one gene per 140 kb. Perhaps the only message learned here is that the gene density across the maize genome varies to a great degree, and large contiguous sequenced regions can begin to capture the true diversity of maize chromosome architecture.In order to characterize large contiguous regions of maize sequence, we identified and sequenced two B73 BAC contigs from the centromeric region of chromosome 3. These contigs of 961 and 594 kb correspond to contigs 117 and 119, respectively, on maize WebFPC (Wei et al., 2007) and span regions associated with the rf1 (for restorer of fertility1) locus for Texas (T) cytoplasmic male sterility (cmsT; Duvick et al., 1961; Wise et al., 1996). As a foundation for the isolation of the Rf1 locus, four rf1 male-sterile mutants were recovered from a screen of 123,500 flowering plants (Wise et al., 1996). A 5.5-kb Mu1-hybridizing EcoRI restriction fragment was identified that cosegregated with the rf1-m3207 allele. Sequences from this fragment were hybridized to a Rf1 cDNA library, and probes designed from the identified cDNA, p6140-1 (Wise et al., 1999), were found to cosegregate with the rf1 locus in a recombinant population selected from over 10,000 progeny.Using probes designed off the 5.5-kb cosegregating restriction fragment and the p6140-1 cDNA, we have identified two BAC contigs spanning the rf1 locus. Sixteen BACs were sequenced to completion to provide high-quality finished sequence. Here, we present two methods for computational finishing of highly repetitive grass genomes, which were successfully utilized to close 11 TE-induced gaps. Sixteen nested repeat clusters were found, each spanning as much as 155 kb and containing a variety of LTR retrotransposon types and ages of insertion. Genes are found tightly clustered, showing a density rate of one gene per 16 kb within gene islands. Finally, comparative analysis with rice (Oryza sativa) and sorghum (Sorghum bicolor) shows that while many genes are retained across all three species, genes have both been lost and translocated across the genomes.     

Detection of Mycobacterium immunogenum by real-time quantitative Taqman PCR

A quantitative real-time 5′-nuclease (Taqman) PCR technique was developed to specifically detect Mycobacterium immunogenum. rpoB-specific primers and Taqman probe were evaluated for detection of M. immunogenum DNA extracted from pure cultures and from industrial metal working fluids (MWFs). Specificity was confirmed and the sensitivity of detection of M. immunogenum genomic DNA was shown to be approximately 9 fg (2 cell equivalents). When tested on industrial metal working fluids from the UK and USA from which no M. immunogenum CFU were recovered, the assay detected between 3.4 × 10¹ and 1.9 × 10⁴ cell equivalents (CE) per ml, and increased the detection rate over culture to 37.5% (12 of 32 samples). This assay provides a specific, sensitive and rapid method for the detection of M. immunogenum and is applicable within industry for the early detection of this human pathogen and to the possible prevention of hypersensitivity pneumonitis (HP) in workers.     

An interpretation of Fujita's frequency diagrams in phyllotaxis

Fujita's diagrams in phyllotaxis, showing the frequencies of divergence angles as a function of these angles for low phyllotactic patterns such as (2, 1) and (3, 2), which are approximately normal curves centered at the limitdivergence angle of 137.51°, are shown to be puzzling when compared to results and observations in the field. An analysis of these diagrams is proposed, in the context of Fujita's methodology, of data from other sources, of a mathematical theorem on lattices, and of the contact pressure theory of phyllotaxis.