Dispersion of cell-to-cell uncoupling precedes low K+-induced ventricular fibrillation

We hypothesize that hypokalemia-related electrolyte imbalance linked with abnormal elevation of intracellular free Ca2+ concentration can cause metabolic disturbances and subcellular alterations resulting in intercellular uncoupling, which favor the occurrence of malignant arrhythmias. Langendorff-perfused guinea pig heart (n = 44) was subjected to a standard Tyrode solution (2.8 mmol/l K+) followed by a K+-deficient solution (1.4 mmol/l K+). Bipolar ECG of the left atria and ventricle was continuously monitored and the incidence of ventricular fibrillation was evaluated. Myocardial tissue sampling was performed during stabilization, hypokalemia and at the onset of fibrillation. Enzyme activities of succinic dehydrogenase, glycogen phosphorylase and 5-nucleotidase were determined using in situ catalytic histochemistry. The main gap junction protein, connexin-43, was labeled using mouse monoclonal antibody and FITC conjugated goat antimouse antibody. Ultrastructure was examined by transmission electron microscopy. The free Ca2+ concentration was measured by the indo-1 method in ventricular cell cultures exposed to a K+-free medium. The results showed that sustained ventricular fibrillation appeared within 15-30 min of low K+ perfusion. This was preceded by ectopic activity, episodes of bigeminy and tachycardia. Hypokalemia induced moderate reversible and sporadically irreversible subcellular alterations of cardiomyocytes and impairment of intercellular junctions, which were heterogeneously distributed throughout myocardium. Patchy areas with decreased enzyme activities and diminished immunoreactivity of connexin-43 were found. Furthermore, lack of external K+ was accompanied by an increase of intracellular Ca2+. The prevention of Ca2+ overload by either 1 mmol/l Ni2+ (Na+/Ca2+ inhibitor), 2.5 micromol/l verapamil, 10 micromol/l d-sotalol or 10 micromol/l tedisamil was associated with the protection against fibrillation. The results indicate that hypokalemia induces Ca2+ overload injury and disturbances in intercellular coupling. Dispersion of these changes throughout the myocardium may serve as the basis for microreentry circuits and thus favor fibrillation occurrence.     

Exclusion of the C/D box snoRNA gene cluster HBII-52 from a major role in Prader–Willi syndrome

Prader–Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurogenetic disorders caused by the loss of function of imprinted genes in 15q11–q13. The maternally expressed UBE3A gene is affected in AS. Four protein-encoding genes (MKRN3, MAGEL2, NDN and SNURF-SNRPN) and several small nucleolar (sno) RNA genes (HBII-13, HBII-436, HBII-85, HBII-438A, HBII-438B and HBII-52) are expressed from the paternal chromosome only but their contribution to PWS is unclear. To examine the role of the HBII-52 snoRNA genes, we have reinvestigated an AS family with a submicroscopic deletion spanning UBE3A and flanking sequences. By fine mapping of the centromeric deletion breakpoint in this family, we have found that the deletion affects all of the 47 HBII-52 genes. Since the complete loss of the HBII-52 genes in family members who carry the deletion on their paternal chromosome is not associated with an obvious clinical phenotype, we conclude that HBII-52 snoRNA genes do not play a major role in PWS. However, we cannot exclude the possibility that the loss of HBII-52 has a phenotypic effect when accompanied by the loss of function of other genes in 15q11–q13.Electronic Database Information: accession numbers and URLs for data presented herein are as follows: for PAR-4 (accession number AF019617), deletion junction fragment (L15422): GenBank, ; for Angelman syndrome [MIM105830]: Online Mendelian Inheritance in Man (OMIM),     

Host-Interactive Genes in Amerindian Helicobacter pylori Diverge from Their Old World Homologs and Mediate Inflammatory Responses

Helicobacter pylori is the dominant member of the gastric microbiota and has been associated with an increased risk of gastric cancer and peptic ulcers in adults. H. pylori populations have migrated and diverged with human populations, and health effects vary. Here, we describe the whole genome of the cag-positive strain V225d, cultured from a Venezuelan Piaroa Amerindian subject. To gain insight into the evolution and host adaptation of this bacterium, we undertook comparative H. pylori genomic analyses. A robust multiprotein phylogenetic tree reflects the major human migration out of Africa, across Europe, through Asia, and into the New World, placing Amerindian H. pylori as a particularly close sister group to East Asian H. pylori. In contrast, phylogenetic analysis of the host-interactive genes vacA and cagA shows substantial divergence of Amerindian from Old World forms and indicates new genotypes (e.g., VacA m3) involving these loci. Despite deletions in CagA EPIYA and CRPIA domains, V225d stimulates interleukin-8 secretion and the hummingbird phenotype in AGS cells. However, following a 33-week passage in the mouse stomach, these phenotypes were lost in isolate V225-RE, which had a 15-kb deletion in the cag pathogenicity island that truncated CagA and eliminated some of the type IV secretion system genes. Thus, the unusual V225d cag architecture was fully functional via conserved elements, but the natural deletion of 13 cag pathogenicity island genes and the truncation of CagA impaired the ability to induce inflammation.Helicobacter pylori is a microaerophilic bacterium of the Epsilonproteobacteria that has colonized the stomach since early in human evolution (45) and diverged with ancient human migrations (24, 45, 92). Thus, several major H. pylori populations, such as hpAfrica1, hpEurope, hspEAsia, and hspAmerind, whose names indicate their original geographic associations (45, 51), have been defined. In particular, similarities between the hspAmerind and hspEAsia populations suggest that the first colonizers of the New World brought H. pylori with them (24, 28). With recent mixing of human groups, H. pylori populations are also mixing and competing, with an apparent dominance by the hpEurope population at least in Latin America (19).H. pylori usually does not cause illness, but colonization with strains bearing the cag (cytotoxin-associated gene) pathogenicity island (cag PAI) (3, 7, 25, 52, 57, 61, 63) is associated with an increased risk of noncardia gastric adenocarcinoma and peptic ulcer disease (56, 64). Nonetheless, a high prevalence of cag-positive H. pylori strains occurs concurrently with low gastric cancer rates in Africa (40) and some regions in Latin America, such as the Venezuelan savannas and Amazonas (29, 53). Moreover, clinical and epidemiological data provide evidence for an inverse relationship between H. pylori colonization and the prevalence of certain metabolic disorders, esophageal diseases, asthma and allergic disorders, and acute infectious diseases, as well as a direct relationship with improved nutritional status of rural children (3, 14, 34, 37, 49, 68). That the host interaction with an indigenous gastric microbe provides some health benefits to the host is not unexpected given the well-established role of gastrointestinal microflora in maintaining gastroenteric homeostasis (8).The most thoroughly studied H. pylori proteins that interact with human cells are CagA and VacA. CagA is an effector protein injected into gastric epithelial cells by a type IV secretion system encoded by the cag PAI (10, 12, 15, 83). VacA is initially secreted from the bacterial cell by an autotransporter mechanism (16). Both proteins have multiple effects on host cells. Inside the host cell, phosphorylation of CagA on EPIYA repeats in the phosphotyrosine (PY) region (73) induces cellular elongation known as the hummingbird phenotype (72). CagA may also induce secretion of interleukin-8 (IL-8) (11), a process commonly attributed to NF-κB, and disrupt the barrier function of the tight junctions in polarized epithelial cells, leading to a loss of adhesion (1, 5). Other motifs in the PY region promote phosphorylation-independent effects (79). In addition, cagA may be considered an oncogene (60), since transgenic expression of cagA in mice leads to gastric epithelial hyperplasia through aberrant epithelial cell signaling and gastric carcinogenesis (60, 62). In contrast, VacA is a multifunctional protein with several activities in epithelial and immune cells (16). VacA induces cell vacuolation (43), alters mitochondrial membrane permeability (27, 41, 90), and increases epithelial monolayer permeability. VacA also activates several signal transduction pathways that are important in immune and epithelial cells, including the mitogen-activated protein (MAP) kinase and p38/ATF-2-mediated signal pathways (9, 55).Genomic analysis provides insights into the evolution of H. pylori strains and their relation with their human hosts and may be useful for the development of diagnostic tools and novel therapies. To date, there are six published complete H. pylori genomes, mostly from the hpEurope population (see Table SA1 in the supplemental material). Here, we report the whole genome of a newly characterized hspAmerind strain, V225d, and assess its genetic structure in comparison to those of Old World H. pylori strains through a comprehensive multiprotein phylogenetic analysis, as well as through single-gene examination of cagA and vacA, revealing clues to the evolution and migration of this strain into the New World and the implications for human health. We also present the results of functional and genomic studies using gastric epithelial cells demonstrating that V225d can induce an inflammatory host response, an effect that was lost following passage through the mouse stomach.     

Detection of biological threat agents by immunomagnetic microsphere-based solid phase fluorogenic- and electro-chemiluminescence

This article reviews the recent development of two solid-phase chemiluminescence-based techniques, fluorogenic-chemiluminescence (FCL) and electro-chemiluminescence (ECL) for detection of biological threat agents. Both techniques entail a labeled sandwich immunoassay. The objectives of this work are to develop advanced techniques for sensitive and effective detection of a target analyte, particularly in cases where the analysis includes complex samples containing multiple contaminating factors. Other important considerations in developing such detection techniques include the ease of use, the rapid determination of the results, and system automation for field applications. In FCL, alkaline phosphatase is used as a label and this technique utilizes the dual features of fluorescence and visual color generated upon the presence of the fluorogenic compound, AttoPhos. The assay reaction is determined by measuring the fluorescence. In ECL, the label is a ruthenium-trisbipyridal, which is excited to a higher energy state by an electric current-driven redox reaction, and the extent of the reaction is assessed via photon emission. Both techniques depend upon the magnetic separation technique as a means to isolate the target immunological agents from the sample for analysis. This magnetic capture system allows for a reaction to occur on the electron effective-transfer zone in the ECL and also provides the reaction site for the labeled sandwich in the FCL. Comparative studies of these two techniques for detection of biological threat agents have been performed and the advantages of using magnetic microspheres versus conventional solid-phase matrices are discussed.     

Antibiotic innovation may contribute to slowing the dissemination of multiresistant Streptococcus pneumoniae: the example of ketolides

Background

Despite increasingly frequent bacterial resistance to antibiotics, antibacterial innovation is rare. Ketolides constitute one of the very few new antibiotic classes active against Streptococcus pneumoniae developed during the last 25 years. Their mechanism of action resembles that of macrolides, but they are unaffected by common resistance mechanisms. However, cross-resistance to ketolides has been observed in some macrolide-resistant strains. We examined how new antibiotic exposure may affect overall pneumococcal resistance patterns in the population. The aims of this study were to assess the potential dissemination of newly emerged resistances and to control the selection of strains already multiresistant to existing antimicrobials.

Methodology/Principal Findings

We developed an age-structured population model for S. pneumoniae transmission in a human community exposed to heptavalent vaccine, and β-lactams, macrolides and ketolides. The dynamics of intra-individual selection of resistant strains under antibiotic exposure and interindividual transmission were simulated, with antibiotic-specific resistance mechanisms defining the path to co-resistances and cross-resistances, and parameters concerning the French situation. Results of this simulation study suggest that new antibiotic consumption could markedly slow the diffusion of multiresistant strains. Wider use was associated with slower progression of multiresistance. When ketolides were prescribed to all ages, resistance to them reached 10% after >15 years, while it took >40 years when they were prescribed only to adults. In the scenario according to which new antibiotics totally replaced former antimicrobials, the β-lactam resistance rate was limited at 70%.

Conclusions

In a context of widespread vaccination and rational use of antibiotics, innovative antibiotic, prescribed to all age groups, may have an added impact on multiresistant-strain dissemination in the population.     

Regulatory signals for endothelial podosome formation

Podosomes are punctate actin-rich adhesion structures which spontaneously form in cells of the myelomonocytic lineage. Their formation is dependent on Src and RhoGTPases. Recently, podosomes have also been described in vascular cells. These podosomes differ from the former by the fact that they are inducible. In endothelial cells, such a signal can be provided by either constitutively active Cdc42, the PKC activator PMA or TGFbeta, depending on the model. Consequently, other regulatory pathways have been reported to contribute to podosome formation. To get more insight into the mechanisms by which podosomes form in endothelial cells, we have explored the respective contribution of signal transducers such as Cdc42-related GTPases, Smads and PKCs in three endothelial cell models. Results presented demonstrate that, in addition to Cdc42, TC10 and TCL GTPases can also promote podosome formation in endothelial cells. We also show that PKCalpha can be either necessary or entirely dispensable, depending on the cell model. In contrast, PKCdelta is essential for podosome formation in endothelial cells but not smooth muscle cells. Finally, although podosomes vary very little in their molecular composition, the signalling pathways involved in their assembly appear very diverse.     

Symbiosis-independent and symbiosis-incompetent mutants of Bdellovibrio bacteriovorus 109J.

Symbiosis-independent (Sin) mutants were isolated from the symbiosis-dependent and symbiosis-competent (Sdcomp+) Bdellovibrio bacteriovorus 109J. Independently isolated Sin mutants were examined for their symbiosis competence and most were found to be comp+. Bdellovibrios comp- were selected from the Sincomp+ mutants. The Sincomp+ bdellovibrios are always at a selective disadvantage, either against Sincomp- bdellovibrios (in organic medium) or against Sdcomp+ bdellovibrios (in buffer with Escherichia coli cells).