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.Bacteria of the genus Bordetella are important pathogens that cause respiratory disease in humans and animals. B. pertussis is the causative agent of whooping cough (8), which is responsible for up to 500,000 annual deaths worldwide in unvaccinated populations (52) and is increasing in incidence in some countries with established vaccination programs, including the United States (e.g., see references 15, 30, and 38). Although most Bordetella species can cause similar disease in the upper respiratory tract, their host specificities vary dramatically. B. pertussis infects only humans; B. parapertussis strains can be classified in two groups, one of which infects only humans and one of which infects only sheep (16,17,25); B. bronchiseptica causes respiratory infections in a wide variety of mammals and birds but only rarely in humans (22,41,51).These three Bordetella species are closely related at the nucleotide sequence level (4, 24), and their relatedness has been further established using a variety of common molecular strain typing techniques (reviewed in reference 29). Phylogenetic analyses of the genus Bordetella, using pulsed-field gel electrophoresis, multilocus enzyme electrophoresis (MLEE), and IS typing data, suggested that B. pertussis, human-derived B. parapertussis, and sheep-derived B. parapertussis arose independently from B. bronchiseptica-like ancestors (46, 47). Comparative genome sequencing of a representative strain of each species (33) confirmed that nucleotide sequence similarity is very high in conserved regions of the genome but demonstrated that B. pertussis and B. parapertussis evolved by genome decay from a B. bronchiseptica-like ancestor.Although a number of critical conserved virulence mechanisms have been identified in the bor...

Primary cilia are mechanosensors for fluid shear stress, and are involved in a number of syndromes and congenital anomalies. We identified endothelial cilia in areas of low shear stress in the embryonic heart. The objective of the present study was to demonstrate the role of primary cilia in mechanosensing. Ciliated embryonic endothelial cells were cultured from the heart, and non-ciliated cells from the arteries. Nonciliated cells that were subjected to fluid shear stress showed significantly less induction of the shear marker Krü ppel-Like Factor-2, as compared to ciliated cells. In addition, ciliated cells from which the cilia were chemically removed show a similar decrease in flow response. This shows that primary cilia sensitize endothelial cells for fluid shear stress. In addition, we targeted and stabilized the connection of the cilium to the cytoplasm by treatment with Colchicine and Taxol/Paclitaxel, respectively, and show that microtubular integrity is essential to sense shear stress. Developmental Dynamics 237:725-735, 2008.

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.

Endothelial cells (EC) translate biomechanical forces into functional and phenotypic responses that play important roles in cardiac development. Specifically, EC in areas of high shear stress, i.e., in the cardiac outflow tract and atrioventricular canal, are characterized by high expression of Krü ppel-like factor 2 (Klf2) and by transforming growth factor-beta (Tgfb)-driven endothelial-to-mesenchymal transition. Extraembryonic venous obstruction (venous clip model) results in congenital heart malformations, and venous clip-induced alterations in shear stress-related gene expression are suggestive for an increase in cardiac shear stress. Here, we study the effects of shear stress on Klf2 expression and Tgfb-associated signaling in embryonic EC in vivo using the venous clip model and in vitro by subjecting cultured EC to fluid flow. Cellular responses were assessed by analysis of Klf2, Tgfb ligands, and their downstream signaling targets. Results show that, in embryonic EC, shear stress activates Tgfb/Alk5 signaling and that induction of Klf2 is an Alk5 dependent process. Developmental Dynamics 240: [1670][1671][1672][1673][1674][1675][1676][1677][1678][1679][1680] 2011. V C 2011 Wiley-Liss, Inc.Key words: shear stress; cardiac cushions; endothelium; Klf2; Tgfb; Alk5 Accepted 14 April 2011 INTRODUCTIONBlood flow is a decisive factor guiding proper cardiovascular development and preventing vascular pathology. Biomechanical forces acting upon the vessel wall include flow-induced shear stress and pressure related cyclic stretch. Shear stress modulates endothelial structure and function and results in an accurate regulation of endothelial gene expression in vitro (Dekker et al., 2002) and in vivo (Groenendijk et al., 2004;Dekker et al., 2005). Several animal models show that congenital malformations can result from disturbed blood flow (Hogers et al., 1997;Sedmera et al., 1999;Tobita et al., 2002;Hove et al., 2003;Dealmeida et al., 2007;Butcher et al., 2007). The large and rapid changes in volume and geometry of cardiac compartments during development are translated into local changes in shear stress and concomitant gene expression patterns of, e.g., Krü ppel like factor 2 (KLF2) (Groenendijk et al., 2004).KLF2 is a shear responsive transcription factor essential in establishing and maintaining endothelial function by regulating the expression of many genes (Dekker et al., 2002(Dekker et al., , 2006SenBanerjee et al., 2004;Boon and Horrevoets, 2009). Exposure of adult endothelium to high and pulsatile shear stress causes an increase in KLF2 expression (Dekker et al., 2002;Wang et al., 2006), which induces a quiescent and atheroprotective phenotype, in part through inhibition of transforming growth factor-beta (TGFb) signaling (Boon et al., 2007). The increase of KLF2 protein levels under shear stress is mediated by two mechanisms, i.e., activation of the KLF2 promoter and stabilization of KLF2 mRNA (Dekker et al., 2002;van Thienen et al., 2006). Myocyte enhancer binding factor 2C (Mef2C) is one of the transcriptiona...

Purpose: The combination of radiotherapy and good quality surgery reduces local recurrence rate for rectal cancer patients. This study assesses the prognostic value of both intrinsic and radiotherapy-induced apoptosis and evaluates the relevance of radiotherapy for outcome of rectal cancer patients. Experimental Design: Tumor samples (1,198) were available from the Dutch Total Mesorectal Excision trial, in which rectal cancer patients were treated with standardized surgery and randomized for preoperative short-term radiotherapy or not. Tumor samples were obtained at time of surgery. Tissue microarrays were constructed and stained with the active caspase-specific M30 antibody to determine the amount of apoptotic epithelial tumor cells. Results: Nonirradiated patients with a negative circumferential margin displaying lower than median levels of apoptosis developed more local recurrences (10.5% versus 6.1%; P = 0.06) and more rapidly after surgery than patients with high intrinsic apoptosis in their tumors (median time to recurrence, 13.0 versus 21.3 months; P = 0.04). In multivariate analysis, intrinsic apoptosis was an independent predictor for the development of local recurrences (hazard ratio, 2.0; P = 0.05). Radiotherapy increased apoptosis level (11 versus 23 apoptotic cells/mm 2 tumor epithelium; P < 0.001), but this apoptosis did not influence patients' prognosis.Conclusions: Rectal cancer patients with low intrinsic apoptosis will benefit from radiotherapy with respect to the development of local recurrences. Because apoptosis is an inherent characteristic of tumors, patients who do not need radiotherapy may be selected based on the apoptotic index of the primary tumor.Local recurrences are a serious problem in the treatment of rectal cancer. To improve local control, total mesorectal excision (TME) surgery has been introduced (1). In addition to TME, preoperative radiotherapy resulted in a further reduction of local recurrences (2) and has been accepted recently as standard treatment for rectal cancer patients in the Netherlands.Although radiotherapy can prevent local recurrences in some patients, the majority of patients will not develop a recurrence without radiotherapy. Moreover, radiotherapy has several negative side effects (3, 4), emphasizing the need for prognostic indicators to select patients most likely benefiting from radiotherapy. Because radiotherapy is known to induce DNA damage, which can result in apoptotic cell death, the better local control observed after radiotherapy might result from the induction of apoptosis. During tumor development, tumor cells can be triggered by lymphocytes of the patient's immune system or other stress factors, such as growth factor deprivation, to undergo apoptosis (5, 6). However, tumor cells that acquired multiple antiapoptotic mutations fail to undergo apoptosis, resulting in selective outgrowth of these cells. The level of apoptosis, both intrinsic and after radiotherapy, might therefore have a prognostic value for the clinical outcome of rectal ...