Polycistronic lentivirus induced pluripotent stem cells from skin biopsies after long term storage, blood outgrowth endothelial cells and cells from milk teeth.

The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue, but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus, fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies, but that of BOECs was lower. In terms of invasiveness of biopsy sampling, biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs, but where non-invasive procedures are required (e.g., for children and minors) dental pulp cells from milk teeth represent a valuable alternative.

Sequence variability in the first internal transcribed spacer region within and among Cyclospora species is consistent with polyparasitism.

Cyclospora cayetanensis is a coccidian parasite which causes severe gastroenteritis in humans. Molecular information on this newly emerging pathogen is scarce. Our objectives were to assess genetic variation within and between human-associated C. cayetanensis and baboon-associated Cyclospora papionis by examining the internal transcribed spacer (ITS) region of the ribosomal RNA operon, and to develop an efficient polymerase chain reaction- (PCR)-based method to distinguish C. cayetanensis from other closely related organisms. For these purposes, we studied C. cayetanensis ITS-1 nucleotide variability in 24 human faecal samples from five geographic locations and C. papionis ITS-1 variability in four baboon faecal samples from Tanzania. In addition, a continuous sequence encompassing ITS-1, 5.8S rDNA and ITS-2 was determined from two C. cayetanensis samples. The results indicate that C. cayetanensis and C. papionis have distinct ITS-1 sequences, but identical 5.8S rDNA sequences. ITS-1 is highly variable within and between samples, but variability does not correlate with geographic origin of the samples. Despite this variability, conserved species-specific ITS-1 sequences were identified and a single-round, C. cayetanensis-specific PCR-based assay with a sensitivity of one to ten oocysts was developed. This consistent and remarkable diversity among Cyclospora spp. ITS-1 sequences argues for polyparasitism and simultaneous transmission of multiple strains.

Analysis of Moraxella catarrhalis by DNA typing: evidence for a distinct subpopulation associated with virulence traits.

Two DNA typing methods, probe-generated restriction fragment length polymorphism analysis and single-adapter amplified fragment length polymorphism analysis, were used to study the genetic relationships among 90 Moraxella catarrhalis strains. Both methods were found to be highly concordant, generating a dendrogram with 2 main branches. The division of the M. catarrhalis population into 2 subspecies was supported by analysis of the 16S rRNA sequences. Both beta-lactamase-positive and beta-lactamase-negative strains were found in all main branches, suggesting horizontal transfer of the beta-lactamase gene. In contrast, 2 virulence traits, complement resistance and adherence to epithelial cells, were strongly associated with 1 of the 2 subspecies. The branch depth suggested that complement-resistant adherent strains diverged from a common ancestor more recently than did complement-sensitive nonadherent strains. These findings suggest the existence of subpopulations of M. catarrhalis that differ in virulence, and they may have implications for vaccine development.

Bordetella species are distinguished by patterns of substantial gene loss and host adaptation.

Pathogens of the bacterial genus Bordetella cause respiratory disease in humans and animals. Although virulence and host specificity vary across the genus, the genetic determinants of this diversity remain unidentified. To identify genes that may underlie key phenotypic differences between these species and clarify their evolutionary relationships, we performed a comparative analysis of genome content in 42 Bordetella strains by hybridization of genomic DNA to a microarray representing the genomes of three Bordetella species and by subtractive hybridization. Here we show that B. pertussis and B. parapertussis are predominantly differentiated from B. bronchiseptica by large, species-specific regions of difference, many of which encode or direct synthesis of surface structures, including lipopolysaccharide O antigen, which may be important determinants of host specificity. The species also exhibit sequence diversity at a number of surface protein-encoding loci, including the fimbrial major subunit gene, fim2. Gene loss, rather than gene acquisition, accompanied by the proliferation of transposons, has played a fundamental role in the evolution of the pathogenic bordetellae and may represent a conserved evolutionary mechanism among other groups of microbial pathogens.