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Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron–exon boundary, although the distance between these SNPs and the intron–exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function., Author Summary Genetic variation, through its effects on gene expression, influences many aspects of the human phenotype. Understanding the impact of genetic variation on human disease risk has become a major goal for biomedical research and has the potential of revealing both novel disease mechanisms and novel functional elements controlling gene expression. Recent large-scale studies have suggested that a relatively high proportion of human genes show allele-specific variation in expression. Effects of common DNA polymorphisms on mRNA splicing are less well studied. Variation in splicing patterns is known to be tissue specific, and for a small number of genes has been shown to vary among individuals. What is not known is whether allele-specific splicing events are an important mechanism by which common genetic variation affects gene expression. In this study we show that allele-specific alternative splicing was observed in six out of 70 exon-skipping events. Sequence analysis of the relevant splice sites and of the regions surrounding single nucleotide polymorphisms correlated with the splicing events failed to identify any predictive bioinformatic signals. A genome-wide study of allele-specific splicing, using an experimental rather than a bioinformatic approach, is now required.

A multilocus sequence typing (MLST) system was developed for group B streptococcus (GBS). The system was used to characterize a collection ( n = 152) of globally and ecologically diverse human strains of GBS that included representatives of capsular serotypes Ia, Ib, II, III, V, VI, and VIII. Fragments (459 to 519 bp) of seven housekeeping genes were amplified by PCR for each strain and sequenced. The combination of alleles at the seven loci provided an allelic profile or sequence type (ST) for each strain. A subset of the strains were characterized by restriction digest patterning, and these results were highly congruent with those obtained with MLST. There were 29 STs, but 66% of isolates were assigned to four major STs. ST-1 and ST-19 were significantly associated with asymptomatic carriage, whereas ST-23 included both carried and invasive strains. All 44 isolates of ST-17 were serotype III clones, and this ST appeared to define a homogeneous clone that was strongly associated with neonatal invasive infections. The finding that isolates with different capsular serotypes had the same ST suggests that recombination occurs at the capsular locus. A web site for GBS MLST was set up and can be accessed at http://sagalactiae.mlst.net. The GBS MLST system offers investigators a valuable typing tool that will promote further investigation of the population biology of this organism.

The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host-parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria.

The identification of clones within bacterial populations is often taken as evidence for a low rate of recombination, but the validity of this inference is rarely examined. We have used statistical tests of congruence between gene trees to examine the extent and significance of recombination in six bacterial pathogens. For Neisseria meningitidis , Streptococcus pneumoniae , Streptococcus pyogenes, and Staphylococcus aureus , the congruence between the maximum likelihood trees reconstructed using seven house-keeping genes was in most cases no better than that between each tree and trees of random topology. The lack of congruence between gene trees in these four species, which include both naturally transformable and nontransformable species, is in three cases supported by high ratios of recombination to point mutation during clonal diversification (estimates of this parameter were not possible for Strep. pyogenes ). In contrast, gene trees constructed for Hemophilus influenzae and pathogenic isolates of Escherichia coli showed a higher degree of congruence, suggesting lower rates of recombination. The impact of recombination therefore varies between bacterial species but in many species is sufficient to obliterate the phylogenetic signal in gene trees.

Background: Currently, more than 600,000 immigrants enter the United States each year from countries where intestinal parasites are endemic. At entry persons with parasitic infections may be asymptomatic, and stool examinations are not a sensitive method of screening for parasitosis. Albendazole is a new, broad-spectrum antiparasitic drug, which was approved recently by the Food and Drug Administration. International trials have shown albendazole to be safe and effective in eradicating many parasites. In the United States there is now disagreement about whether to screen all immigrants for parasites, treat all immigrants presumptively, or do nothing unless they have symptoms. Methods: We compared the costs and benefits of no preventive intervention (watchful waiting) with those of universal screening or presumptive treatment with 400 mg of albendazole per day for five days. Those at risk were defined as immigrants to the United States from Asia, the Middle East, sub-Saharan Africa, Eastern Europe, and Latin America and the Caribbean. Cost effectiveness was expressed both in terms of the cost of treatment per disability adjusted life-year (DALY) averted (one DALY is defined as the loss of one year of healthy life to disease)and in terms of the cost per hospitalization averted. Results: As compared with watchful waiting, presumptive treatment of all immigrants at risk for parasitosis would avert at least 870 DALYs, prevent at least 33 deaths and 374 hospitalizations, and save at least $4.2 million per year. As compared with watchful waiting, screening would cost $159,236 per DALY averted. Conclusions: Presumptive administration of albendazole to all immigrants at risk for parasitosis would save lives and money. Universal screening, with treatment of persons with positive stool examinations, would save lives but is less cost effective than presumptive treatment.

Summary SummaryA population dynamic model of schistosome transmission was used to investigate the interaction between drug efficacy and drug price of different brands of praziquantel in determining the cost-effectiveness of school-targeted treatment for Schistosoma mansoni. In this analysis, costs were affected by coverage, drug price and distance travelled, and effectiveness by coverage and drug efficacy. Four effectiveness measures were assessed: the number of infection case-years prevented, heavy infection case-years prevented, hepatomegaly case-years prevented and fibrosis case-years prevented. The interactions between drug efficacy and drug price were complex. In particular, there was a highly nonlinear relationship between drug efficacy and cost-effectiveness, with drugs of low efficacy producing high and variable cost-effectiveness ratios, particularly when other programme costs related to distance travelled were high. The results suggest that given the current price range of praziquantel, a drug with less than a 50% chance of killing the worms is not to be recommended. This has important practical implications for the widespread use of praziquantel, since most international agencies procure praziquantel purely on the basis of price. There is clearly a need for studies which evaluate the efficacy of new brands of praziquantel, and more credence should be given to the use of high efficacy brands, not only in terms of maximizing the cost-effectiveness of the intervention programme, but also in delaying the onset of drug resistance.

Summary: The 32-bit Windows application START is implemented using Visual Basic and C ++ and performs analyses to aid in the investigation of bacterial population structure using multilocus sequence data. These analyses include data summary, lineage assignment, and tests for recombination and selection. Availability: START is available at http://outbreak.ceid.ox. ac.uk/software.htm. Contact: keith.jolley@ceid.ox.ac.uk Multilocus Sequence Typing (MLST) is a nucleotide sequence-based typing method that indexes the variation present in bacterial housekeeping genes, where most of the variation is selectively neutral (Maiden et al., 1998). Internal fragments of seven housekeeping genes, approximately 450–500 bp in length, are sequenced and novel alleles are assigned with arbitrary numbers sequentially to provide an allelic profile of seven integers that defines the Sequence Type (ST) of each isolate. The technique is designed primarily for global or long-term epidemiology and surveillance, and has the advantage over other typing methods, such as genetic fingerprinting, of electronic portability and unambiguous characterization of isolates. MLST schemes have been developed for a range of bacterial pathogens and databases for these organisms can be interrogated at the MLST web-site (http://www.mlst.net/) thus facilitating rapid comparisons of isolates typed using the method. A further advantage of MLST is that it provides large quantities of data that may be analyzed by a number of evolutionary approaches to yield insights into the structure of bacterial populations and the selective pressures which act upon them. With the increasing availability of MLST data, the need for software to describe and analyze datasets has become apparent. Sequence Type Analysis and Recombinational Tests (START) was written to address this need through the inclusion of multiple analytical techniques in an easyto-use and intuitive interface for Windows 95/98/NT/2000 operating systems.

Multi-locus sequence typing (MLST) is a method of typing that facilitates the discrimination of microbial isolates by comparing the sequences of housekeeping gene fragments. The mlstdbNet software enables the implementation of distributed web-accessible MLST databases that can be linked widely over the Internet. The software enables multiple isolate databases to query a single profiles database that contains allelic profile and sequence definitions. This separation enables isolate databases to be established by individual laboratories, each customised to the needs of the particular project and with appropriate access restrictions, while maintaining the benefits of a single definitive source of profile and sequence information. Databases are described by an XML file that is parsed by a Perl CGI script. The software offers a large number of ways to query the databases and to further break down and export the results generated. Additional features can be enabled by installing third-party (freely available) tools. Development of a distributed structure for MLST databases offers scalability and flexibility, allowing participating centres to maintain ownership of their own data, without introducing duplication and data integrity issues.

The dynamics of a virus disease in a perennial plant population are modelled. The population is divided into healthy, latently infected, infectious and post-infectious plants and linked differential equations describe the dynamics of each category. Qualitative analysis of this model shows stable dynamics and threshold conditions for disease persistence. Stable equilibria are reached after several years. The dynamics of the model are highly sensitive to changes in contact rate and infectious period. Disease management by roguing (removal) of infected plants and replanting with healthy ones is investigated. Roguing only in the post-infectious category confers no advantage. At low contact rates, roguing only when plants become infectious is sufficient to eradicate the disease (...)

Haplotype-based techniques are being used to estimate the relative age of alleles--particularly in screening loci for signals of recent positive selection--but does this approach capture even coarse age differences? Using simulations and empirical data from the International HapMap Project, we show that a simple pairwise metric of haplotype homozygosity gives significantly higher mean values for human single-nucleotide-polymorphism alleles that appear to be derived than for those that appear to be ancestral, as determined by comparison with the chimpanzee genome. Our results support the use of haplotype-based techniques, such as extended haplotypic homozygosity, to assess the age of alleles.

International audience; We previously reported a shift in the electrophoretic type (ET) of invasive MenC in Canada from predominantly ET-15 to ET-37 in the post-MenC conjugate vaccine period. This study sought to confirm this trend by examining all culture-confirmed invasive MenC case isolates in Canada in the period from 1 January 2009 to 31 December 2013. Of the 50 MenC isolates, 18 belonged to ET-15, 28 belonged to ET-37 (but not ET-15), and four belonged to other clonal types. Analysis of the serotype and serosubtype antigens, porA and fetA gene sequences provided data to show that invasive MenC belonging to ET-15 and ET-37 were two very different subpopulations within the ST-11 clonal complex. Sequence analysis of the fHbp genes suggested that 12 different types of factor H-binding protein were found among the ET-15 isolates while 86 % of ET-37 isolates were found to have fHbp genes predicted to encode peptide 22. The nadA gene in 12 MenC isolates was disrupted due to IS1301 insertion and 11 of these 12 isolates belonged to ET-15. Ten per cent of the invasive MenC were found to have a frame-shift mutation in their fHbp genes that predicted no fHbp produced. Significant diversity and frame-shift mutations of fHbp genes were found in invasive MenC strains in Canada.

International audience; From 2006 to December 2009, 45 out of the 513 strains isolated from patients with invasive meningococcal disease in Belgium, were identified as Neisseria meningitidis serogroup B, non-serotypeable, subtype P1.14 (B:NT:P1.14). Most cases were geographically clustered in the northern part of the country. Multilocus Sequence Typing and antigen gene sequencing combined with Pulsed-Field Gel electrophoresis were used to investigate this cluster. Molecular typing showed that 39 out of these 45 N. meningitidis strains belonged to the clonal complex cc-269. The presence of the same PorA Variable Regions (VR1-VR2: 22, 14), the FetA allele (F5-1) and the highly similar Pulsed-Field Gel Electrophoresis profiles, supported genetic relatedness for 38 out of these 39 isolates. Retrospective analysis of B:NT:P1.22,14 isolates from 1999 onwards suggested that these strains belonging to the cc-269 complex, first emerged in the Belgian province of West-Flanders in 2004. This study showed that the combination of molecular tools with classical methods enabled reliable outbreak detection as well as a cluster identification.