Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells

Neisseria meningitidis is a human pathogen, which is a major cause of sepsis and meningitis. The bacterium colonizes the upper respiratory tract of approximately 10% of humans where it lives as a commensal. On rare occasions, it crosses the epithelium and reaches the bloodstream causing sepsis. From the bloodstream it translocates the blood-brain barrier, causing meningitis. Although all strains have the potential to cause disease, a subset of them, which belongs to hypervirulent lineages, causes disease more frequently than others. Recently, we described NadA, a novel antigen of N. meningitidis, present in three of the four known hypervirulent lineages. Here we show that NadA is a novel bacterial invasin which, when expressed on the surface of Escherichia coli, promotes adhesion to and invasion into Chang epithelial cells. Deletion of the N-terminal globular domain of recombinant NadA or pronase treatment of human cells abrogated the adhesive phenotype. A hypervirulent strain of N. meningitidis where the nad A gene was inactivated had a reduced ability to adhere to and invade into epithelial cells in vitro. NadA is likely to improve the fitness of N. meningitidis contributing to the increased virulence of strains that belong to the hypervirulent lineages.     

Biotechnology and vaccines: application of functional genomics to Neisseria meningitidis and other bacterial pathogens

Since its introduction, vaccinology has been very effective in preventing infectious diseases. However, in several cases, the conventional approach to identify protective antigens, based on biochemical, immunological and microbiological methods, has failed to deliver successful vaccine candidates against major bacterial pathogens. The recent development of powerful biotechnological tools applied to genome-based approaches has revolutionized vaccine development, biological research and clinical diagnostics. The availability of a genome provides an inclusive virtual catalogue of all the potential antigens from which it is possible to select the molecules that are likely to be more effective. Here, we describe the use of "reverse vaccinology", which has been successful in the identification of potential vaccines candidates against Neisseria meningitidis serogroup B and review the use of functional genomics approaches as DNA microarrays, proteomics and comparative genome analysis for the identification of virulence factors and novel vaccine candidates. In addition, we describe the potential of these powerful technologies in understanding the pathogenesis of various bacteria.     

The role of intravascular ultrasound imaging in vascular brachytherapy

Intracoronary brachytherapy has recently emerged as a new therapy to prevent restenosis. Initial experimental work was achieved in animal models and the results were assessed by histomorphometry. Initial clinical trials used angiography to guide dosimetry and to assess efficacy. Intravascular ultrasound (IVUS) permits tomographic examination of the vessel wall, elucidating the true morphology of the lumen and transmural components, which cannot be investigated on the lumenogram obtained by angiography. This paper reviews the use of IVUS in the clinical studies of brachytherapy conducted to date. IVUS allows clinicians to make a thorough assessment of the remodeling of the vessel and appears to have a major role to play in facilitating understanding of the underlying mechanisms of action in this emerging field. The authors propose that state-of-the-art IVUS techniques should be employed to further knowledge of the mechanisms of action of brachytherapy in atherosclerotic human coronary arteries.     

Automated Entire Thrombus Density Measurements for Robust and Comprehensive Thrombus Characterization in Patients with Acute Ischemic Stroke

Background and Purpose

In acute ischemic stroke (AIS) management, CT-based thrombus density has been associated with treatment success. However, currently used thrombus measurements are prone to inter-observer variability and oversimplify the heterogeneous thrombus composition. Our aim was first to introduce an automated method to assess the entire thrombus density and then to compare the measured entire thrombus density with respect to current standard manual measurements.

Materials and Method

In 135 AIS patients, the density distribution of the entire thrombus was determined. Density distributions were described using medians, interquartile ranges (IQR), kurtosis, and skewedness. Differences between the median of entire thrombus measurements and commonly applied manual measurements using 3 regions of interest were determined using linear regression.

Results

Density distributions varied considerably with medians ranging from 20.0 to 62.8 HU and IQRs ranging from 9.3 to 55.8 HU. The average median of the thrombus density distributions (43.5 ± 10.2 HU) was lower than the manual assessment (49.6 ± 8.0 HU) (p<0.05). The difference between manual measurements and median density of entire thrombus decreased with increasing density (r = 0.64; p<0.05), revealing relatively higher manual measurements for low density thrombi such that manual density measurement tend overestimates the real thrombus density.

Conclusions

Automatic measurements of the full thrombus expose a wide variety of thrombi density distribution, which is not grasped with currently used manual measurement. Furthermore, discrimination of low and high density thrombi is improved with the automated method.     

Improving model construction of profile HMMs for remote homology detection through structural alignment

Background  

Remote homology detection is a challenging problem in Bioinformatics. Arguably, profile Hidden Markov Models (pHMMs) are one of the most successful approaches in addressing this important problem. pHMM packages present a relatively small computational cost, and perform particularly well at recognizing remote homologies. This raises the question of whether structural alignments could impact the performance of pHMMs trained from proteins in the Twilight Zone, as structural alignments are often more accurate than sequence alignments at identifying motifs and functional residues. Next, we assess the impact of using structural alignments in pHMM performance.     

Loss of Eph-receptor expression correlates with loss of cell adhesion and chondrogenic capacity in Hoxa13 mutant limbs.

Mesenchymal patterning is an active process whereby genetic commands coordinate cell adhesion, sorting and condensation, and thereby direct the formation of morphological structures. In mice that lack the Hoxa13 gene, the mesenchymal condensations that form the autopod skeletal elements are poorly resolved, resulting in missing digit, carpal and tarsal elements. In addition, mesenchymal and endothelial cell layers of the umbilical arteries (UAs) are disorganized, resulting in their stenosis and in embryonic death. To further investigate the role of Hoxa13 in these phenotypes, we generated a loss-of-function allele in which the GFP gene was targeted into the Hoxa13 locus. This allele allowed FACS isolation of mesenchymal cells from Hoxa13 heterozygous and mutant homozygous limb buds. Hoxa13(GFP) expressing mesenchymal cells from Hoxa13 mutant homozygous embryos are defective in forming chondrogenic condensations in vitro. Analysis of pro-adhesion molecules in the autopod of Hoxa13 mutants revealed a marked reduction in EphA7 expression in affected digits, as well as in micromass cell cultures prepared from mutant mesenchymal cells. Finally, antibody blocking of the EphA7 extracellular domain severely inhibits the capacity of Hoxa13(GFP) heterozygous cells to condense and form chondrogenic nodules in vitro, which is consistent with the hypothesis that reduction in EphA7 expression affects the capacity of Hoxa13(-/-) mesenchymal cells to form chondrogenic condensations in vivo and in vitro. EphA7 and EphA4 expression were also decreased in the mesenchymal and endothelial cells that form the umbilical arteries in Hoxa13 mutant homozygous embryos. These results suggest that an important role for Hoxa13 during limb and UA development is to regulate genes whose products are required for mesenchymal cell adhesion, sorting and boundary formation.     

Normal limb development in conditional mutants of Fgf4

Fibroblast growth factors (FGFs) mediate multiple developmental signals in vertebrates. Several of these factors are expressed in limb bud structures that direct patterning of the limb. FGF4 is produced in the apical ectodermal ridge (AER) where it is hypothesized to provide mitogenic and morphogenic signals to the underlying mesenchyme that regulate normal limb development. Mutation of this gene in the germline of mice results in early embryonic lethality, preventing subsequent evaluation of Fgf4 function in the AER. A conditional mutant of Fgf4, based on site-specific Cre/loxP-mediated excision of the gene, allowed us to bypass embryonic lethality and directly test the role of FGF4 during limb development in living murine embryos. This conditional mutation was designed so that concomitant with inactivation of the Fgf4 gene by excision of all Fgf4-coding sequences, a reporter gene was activated in Fgf4-expressing cells, allowing assessment of the site-specific recombination reaction. Although a large body of evidence led us to predict that ablation of Fgf4 gene function in the AER of developing mice would result in abnormal limb outgrowth and patterning, we found that Fgf4 conditional mutants had normal limbs. Furthermore, expression patterns of Shh, Bmp2, Fgf8 and Fgf10 were normal in the limb buds of the conditional mutants. These findings indicate that the previously proposed FGF4-SHH feedback loop is not essential for coordination of murine limb outgrowth and patterning. We suggest that some of the roles currently attributed to FGF4 during early vertebrate limb development may be performed by other AER factors in vivo.     

Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells

We mutated, by gene targeting, the endogenous hypoxanthine phosphoribosyl transferase (HPRT) gene in mouse embryo-derived stem (ES) cells. A specialized construct of the neomycin resistance (neor) gene was introduced into an exon of a cloned fragment of the Hprt gene and used to transfect ES cells. Among the G418r colonies, 1/1000 were also resistant to the base analog 6-thioguanine (6-TG). The G418r, 6-TGr cells were all shown to be Hprt- as the result of homologous recombination with the exogenous, neor-containing, Hprt sequences. We have compared the gene-targeting efficiencies of two classes of neor-Hprt recombinant vectors: those that replace the endogenous sequence with the exogenous sequence and those that insert the exogenous sequence into the endogenous sequence. The targeting efficiencies of both classes of vectors are strongly dependent upon the extent of homology between exogenous and endogenous sequences. The protocol described herein should be useful for targeting mutations into any gene.