Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the "Beijing" sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive.

SiRNA inhibits replication of Langat virus, a member of the tick-borne encephalitis virus complex in organotypic rat brain slices

Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000-3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3' untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro.

Parallel evolution of facial stripe patterns in the Neolamprologus brichardilpulcher species complex endemic to Lake Tanganyika

Colour pattern diversity can be due to random processes or to natural or sexual selection. Consequently, similarities in colour patterns are not always correlated with common ancestry, but may result from convergent evolution under shared selection pressures or drift. Neolamprologus brichardi and Neolamprologus pulcher have been described as two distinct species based on differences in the arrangement of two dark bars on the operculum. Our study uses DNA sequences of the mitochondrial control region to show that relatedness of haplotypes disagrees with species assignment based on head colour pattern. This suggests repeated parallel evolution of particular stripe patterns. The complete lack of shared haplotypes between populations of the same or different phenotypes reflects strong philopatric behaviour, possibly induced by the cooperative breeding mode in which offspring remain in their natal territory and serve as helpers until they disperse to nearby territories or take over a breeding position. Concordant phylogeographic patterns between N. brichardi/N. pulcher populations and other rock-dwelling cichlids suggest that the same colonization routes have been taken by sympatric species and that these routes were affected by lake level fluctuations in the past. (C) 2007 Elsevier Inc. All rights reserved.

Classification of the taxon 2 and taxon 3 complex of Bisgaard within Gallibacterium and description of Gallibacterium melopsittaci sp. nov., Gallibacterium trehalosifermentans sp. nov. and Gallibacterium salpingitidis sp. nov

This investigation was based on 23 isolates from several European countries collected over the past 30 years, and included characterization of all isolates. Published data on amplified fragment length polymorphism typing of isolates representing all biovars as well as protein profiles were used to select strains that were then further characterized by polyamine profiling and sequencing of 16S rRNA, infB, rpoB and recN genes. Comparison of 16S rRNA gene sequences revealed a monophyletic group within the avian 16S rRNA group of the Pasteurellaceae, which currently includes the genera Avibacterium, Gallibacterium and Volucribacter. Five monophyletic subgroups related to Gallibacterium anatis were recognized by 16S rRNA, rpoB, infB and recN gene sequence comparisons. Whole-genome similarity between strains of the five subgroups and the type strain of G. anatis calculated from recN sequences allowed us to classify them within the genus Gallibacterium. In addition, phenotypic data including biochemical traits, protein profiling and polyamine patterns clearly indicated that these taxa are related. Major phenotypic diversity was observed for 16S rRNA gene sequence groups. Furthermore, comparison of whole-genome similarities, phenotypic data and published data on amplified fragment length polymorphism and protein profiling revealed that each of the five groups present unique properties that allow the proposal of three novel species of Gallibacterium, for which we propose the names Gallibacterium melopsittaci sp. nov. (type strain F450(T) =CCUG 36331(T) =CCM 7538(T)), Gallibacterium trehalosifermentans sp. nov. (type strain 52/S3/90(T) =CCUG 55631(T) =CCM 7539(T)) and Gallibacterium salpingitidis sp. nov. (type strain F150(T) =CCUG 15564(T) =CCUG 36325(T) =NCTC 11414(T)), a novel genomospecies 3 of Gallibacterium and an unnamed taxon (group V). An emended description of the genus Gallibacterium is also presented.

PCR identification of HIV-1 DNA sequences in brain tissue of patients with AIDS encephalopathy.

We analyzed brain tissue from 39 patients for the presence of proviral HIV-1 sequences, using the polymerase chain reaction (PCR) for the amplification of segments of the viral LTR and gag genes. A novel primer extension procedure allowed the detection of a single HIV-1 copy in 1 micrograms DNA. We detected proviral HIV-1 DNA in 16 of 25 brain samples from AIDS patients. Semiquantitative evaluation of the amplified DNAs indicated considerable variation in viral load. Highest levels of proviral DNA were present in brain samples from six patients with clinical evidence of HIV-associated cognitive/motor complex and the histopathologic correlate of HIV leukoencephalopathy or HIV encephalitis. An additional 11 brain samples contained smaller amounts of proviral DNA. In these patients, clinical data were inconclusive regarding the diagnosis of HIV-1 encephalopathy and histopathologically there was no evidence of HIV-1-induced tissue lesions. In nine of 25 seropositive patients with AIDS (36%), brain samples scored negative or did not contain an unequivocal signal indicating the presence of proviral DNA. HIV-1 sequences were not detected in any of 14 control brain samples from HIV-1 seronegative patients. Our data indicate that HIV-1 is present in the central nervous system of the majority (two thirds) of AIDS patients and that the highest levels of proviral DNA in brain tissue are associated with HIV encephalopathy.

Projecting a relevant "next": Instruction sequences in driving lessons

Car interaction and the organisation of multi-activity in cars have become a fertile topic of research within CA and EM (Laurier 2005, Haddington & Keisanen 2009). While previous research has focused exclusively on everyday car rides, in this paper we will analyse a specific kind of car interaction, namely driving lessons. In addition to"driving" and"talking", as the two main parallel activities in everyday car rides (Mondada in press), in driving lessons a central activity is"instructing", that we understand to be a collaborative accomplishment (Sanchez Svensson et al. 2009). Drawing on a corpus of 7 video-recorded driving lessons, we will analyse the sequential organisation of"instruction sequences", i.e. of those actions that are initiated by the driving instructor with a turn projecting the next relevant action to be executed by the learner. Learners carry out next actions in two different ways: a) as"single" actions (e.g. using the indicator); b) as a complex series of overlapping or parallel actions. We will show that"single" actions occur as responses to instructions concerning the learner's command of the car, while complex actions occur when the instructors formulate direction indications. The aims of our analyses are twofold. Firstly, we will analyse how instruction sequences are fitted to the emerging contingencies of the car ride (movement in space, changing environment): we will show that a) the turn format of the instruction initiation displays the degree of"urgency" of the requested action; b) learners have the possibility to start the relevant"next" before the instruction initiation comes to completion. Secondly, we will focus on those"seconds" that the driving instructor treats as problematic by initiating a repair sequence (e.g. an improper use of the indicator). Our research contributes to the discussion about the multimodal resources that participants can employ to fulfil a projected action. In addition, it offers insights in a hitherto scarcely investigated topic, namely the organisation of instructions and the ecology of apprenticeship. References HADDINGTON, P. & KEISANEN, T. (2009) Location, mobility and the body as resources in selecting a route. Journal of Pragmatics 41 (10), 1938-1961. LAURIER, Eric (2005): Searching for a parking space. Intellectica 41-42/2-3: 101-116. MONDADA, Lorenza (in press). Talking and driving: multi-activity in the car. Semiotica. SANCHEZ SVENSSON, M. et al. (2009) "Embedding instruction in practice: contingency and collaboration during surgical training", Sociology of Health & Illness, 31/6: 889-906.

Echinococcus P29 antigen: molecular characterization and implication on post-surgery follow-up of CE patients infected with different species of the Echinococcus granulosus complex.

The protein P29 is a potential serological marker for post-treatment monitoring of cystic echinococcosis (CE) especially in young patients. We now have demonstrated that P29 is encoded in the Echinococcus genus by a single gene consisting of 7 exons spanning 1.2 kb of DNA. Variability of the p29 gene at inter- and intra-species level was assessed with 50 cDNA and 280 genomic DNA clones isolated from different E. granulosus s.l. isolates (E. granulosus sensu stricto (G1), E. equinus (G4), E. ortleppi (G5), E. canadensis (G6), E. canadensis (G7) and E. canadensis (G10)) as well as four E. multilocularis isolates. Scarce interspecies polymorphism at the p29 locus was observed and affected predominantly E. granulosus s.s. (G1), where we identified two alleles (A1 and A2) coding for identical P29 proteins and yielding in three genotypes (A1/A1, A2/A2 and A1/A2). Genotypic frequencies expected under Hardy-Weinberg equilibrium revealed a high rate of heterozygosity (47%) that strongly supports the hypothesis that E. granulosus s.s. (G1) is predominantly outbreeding. Comparative sequence analyses of the complete p29 gene showed that phylogenetic relationships within the genus Echinococcus were in agreement with those of previous nuclear gene studies. At the protein level, the deduced P29 amino acid (AA) sequences exhibited a high level of conservation, ranging from 97.9% AA sequence identity among the whole E. granulosus s.l. group to 99.58% identity among E. multilocularis isolates. We showed that P29 proteins of these two species differ by three AA substitutions without implication for antigenicity. In Western-blot analyses, serum antibodies from a human CE patient infected with E. canadensis (G6) strongly reacted with recombinant P29 from E. granulosus s.s. (G1) (recEg(G1)P29). In the same line, human anti-Eg(G1)P29 antibodies bound to recEcnd(G6)P29. Thus, minor AA sequence variations appear not to impair the prognostic serological use of P29.

Fine-mapping and mutation analysis of TRPM1: a candidate gene for leopard complex (LP) spotting and congenital stationary night blindness in horses.

Leopard Complex spotting occurs in several breeds of horses and is caused by an incompletely dominant allele (LP). Homozygosity for LP is also associated with congenital stationary night blindness (CSNB) in Appaloosa horses. Previously, LP was mapped to a 6 cm region on ECA1 containing the candidate gene TRPM1 (Transient Receptor Potential Cation Channel, Subfamily M, Member 1) and decreased expression of this gene, measured by qRT-PCR, was identified as the likely cause of both spotting and ocular phenotypes. This study describes investigations for a mutation causing or associated with the Leopard Complex and CSNB phenotype in horses. Re-sequencing of the gene and associated splice sites within the 105 624 bp genomic region of TRPM1 led to the discovery of 18 SNPs. Most of the SNPs did not have a predictive value for the presence of LP. However, one SNP (ECA1:108,249,293 C>T) found within intron 11 had a strong (P < 0.0005), but not complete, association with LP and CSNB and thus is a good marker but unlikely to be causative. To further localize the association, 70 SNPs spanning over two Mb including the TRPM1 gene were genotyped in 192 horses from three different breeds segregating for LP. A single 173 kb haplotype associated with LP and CSNB (ECA1: 108,197,355- 108,370,150) was identified. Illumina sequencing of 300 kb surrounding this haplotype revealed 57 SNP variants. Based on their localization within expressed sequences or regions of high sequence conservation across mammals, six of these SNPs were considered to be the most likely candidate mutations. While the precise function of TRPM1 remains to be elucidated, this work solidifies its functional role in both pigmentation and night vision. Further, this work has identified several potential regulatory elements of the TRPM1 gene that should be investigated further in this and other species.

Biochemical demonstration of complex formation of histone pre-mRNA with U7 small nuclear ribonucleoprotein and hairpin binding factors.

Histone RNA 3' end formation occurs through a specific cleavage reaction that requires, among other things, base-pairing interactions between a conserved spacer element in the pre-mRNA and the minor U7 snRNA present as U7 snRNP. An oligonucleotide complementary to the first 16 nucleotides of U7 RNA can be used to characterize U7 snRNPs from nuclear extracts by native gel electrophoresis. Using similar native gel techniques, we present direct biochemical evidence for a stable association between histone pre-mRNA and U7 snRNPs. Other complexes formed in the nuclear extract are dependent on the 5' cap structure and on the conserved hairpin element of histone pre-mRNA, respectively. However, in contrast to the U7-specific complex, their formation is not required for processing. Comparison of several authentic and mutant histone pre-mRNAs with different spacer sequences demonstrates that the formation and stability of the U7-specific complex closely follows the predicted stability of the potential RNA-RNA hybrid. However, this does not exclude a stabilization of the complex by U7 snRNP structural proteins.

First Staphylococcal Cassette Chromosome mec Containing a mecB-Carrying Gene Complex Independent of Transposon Tn6045 in a Macrococcus caseolyticus Isolate from a Canine Infection.

A methicillin-resistant mecB-positive Macrococcus caseolyticus (strain KM45013) was isolated from the nares of a dog with rhinitis. It contained a novel 39-kb transposon-defective complete mecB-carrying staphylococcal cassette chromosome mec element (SCCmecKM45013). SCCmecKM45013 contained 49 coding sequences (CDSs), was integrated at the 3' end of the chromosomal orfX gene, and was delimited at both ends by imperfect direct repeats functioning as integration site sequences (ISSs). SCCmecKM45013 presented two discontinuous regions of homology (SCCmec coverage of 35%) to the chromosomal and transposon Tn6045-associated SCCmec-like element of M. caseolyticus JCSC7096: (i) the mec gene complex (98.8% identity) and (ii) the ccr-carrying segment (91.8% identity). The mec gene complex, located at the right junction of the cassette, also carried the β-lactamase gene blaZm (mecRm-mecIm-mecB-blaZm). SCCmecKM45013 contained two cassette chromosome recombinase genes, ccrAm2 and ccrBm2, which shared 94.3% and 96.6% DNA identity with those of the SCCmec-like element of JCSC7096 but shared less than 52% DNA identity with the staphylococcal ccrAB and ccrC genes. Three distinct extrachromosomal circularized elements (the entire SCCmecKM45013, ΨSCCmecKM45013 lacking the ccr genes, and SCCKM45013 lacking mecB) flanked by one ISS copy, as well as the chromosomal regions remaining after excision, were detected. An unconventional circularized structure carrying the mecB gene complex was associated with two extensive direct repeat regions, which enclosed two open reading frames (ORFs) (ORF46 and ORF51) flanking the chromosomal mecB-carrying gene complex. This study revealed M. caseolyticus as a potential disease-associated bacterium in dogs and also unveiled an SCCmec element carrying mecB not associated with Tn6045 in the genus Macrococcus.