Microscopy of bacterial translocation during small bowel obstruction and ischemia in vivo – a new animal model

BACKGROUND: Existing animal models provide only indirect information about the pathogenesis of infections caused by indigenous gastrointestinal microflora and the kinetics of bacterial translocation. The aim of this study was to develop a novel animal model to assess bacterial translocation and intestinal barrier function in vivo. METHODS: In anaesthetized male Wistar rats, 0.5 ml of a suspension of green fluorescent protein-transfected E. coli was administered by intraluminal injection in a model of small bowel obstruction. Animals were randomly subjected to non-ischemic or ischemic bowel obstruction. Ischemia was induced by selective clamping of the terminal mesenteric vessels feeding the obstructed bowel loop. Time intervals necessary for translocation of E. coli into the submucosal stroma and the muscularis propria was assessed using intravital microscopy. RESULTS: Bacterial translocation into the submucosa and muscularis propria took a mean of 36 ± 8 min and 80 ± 10 min, respectively, in small bowel obstruction. Intestinal ischemia significantly accelerated bacterial translocation into the submucosa (11 ± 5 min, p < 0.0001) and muscularis (66 ± 7 min; p = 0.004). Green fluorescent protein-transfected E. coli were visible in frozen sections of small bowel, mesentery, liver and spleen taken two hours after E. coli administration. CONCLUSIONS: Intravital microscopy of fluorescent bacteria is a novel approach to study bacterial translocation in vivo. We have applied this technique to define minimal bacterial transit time as a functional parameter of intestinal barrier function

Mining Predicted Essential Genes of Brugia malayi for Nematode Drug Targets

We report results from the first genome-wide application of a rational drug target selection methodology to a metazoan pathogen genome, the completed draft sequence of Brugia malayi, a parasitic nematode responsible for human lymphatic filariasis. More than 1.5 billion people worldwide are at risk of contracting lymphatic filariasis and onchocerciasis, a related filarial disease. Drug treatments for filariasis have not changed significantly in over 20 years, and with the risk of resistance rising, there is an urgent need for the development of new anti-filarial drug therapies. The recent publication of the draft genomic sequence for B. malayi enables a genome-wide search for new drug targets. However, there is no functional genomics data in B. malayi to guide the selection of potential drug targets. To circumvent this problem, we have utilized the free-living model nematode Caenorhabditis elegans as a surrogate for B. malayi. Sequence comparisons between the two genomes allow us to map C. elegans orthologs to B. malayi genes. Using these orthology mappings and by incorporating the extensive genomic and functional genomic data, including genome-wide RNAi screens, that already exist for C. elegans, we identify potentially essential genes in B. malayi. Further incorporation of human host genome sequence data and a custom algorithm for prioritization enables us to collect and rank nearly 600 drug target candidates. Previously identified potential drug targets cluster near the top of our prioritized list, lending credibility to our methodology. Over-represented Gene Ontology terms, predicted InterPro domains, and RNAi phenotypes of C. elegans orthologs associated with the potential target pool are identified. By virtue of the selection procedure, the potential B. malayi drug targets highlight components of key processes in nematode biology such as central metabolism, molting and regulation of gene expression

Dynamics of an impact oscillator near a degenerate graze

We give a complete analysis of low-velocity dynamics close to grazingfor a generic one degree of freedom impact oscillator. This includes nondegenerate (quadratic) grazing and minimallydegenerate (cubic) grazing, corresponding respectively to nondegenerateand degenerate chatter. We also describe the dynamics associatedwith generic one-parameter bifurcation at a more degenerate (quartic)graze, showing in particular how this gives rise to the often-observedhighly convoluted structure in the stable manifolds of chattering orbits.The approach adopted is geometric, using methods from singularity theory

Ene-Diene Transmissive Cycloaddition Reactions with Singlet Oxygen: The Vinylogous Gem Effect and Its Use for Polyoxyfunctionalization of Dienes

The singlet oxygen reactivities and regioselectivities of the model compounds 1b-d were compared with those of the geminal (gem) selectivity model ethyl tiglate (1a). The kinetic cis effect is k(E)/k(Z) = 5.2 for the tiglate/angelate system 1a/1a' without a change in the high gem regioselectivity. Further conjugation to vinyl groups enabled mode-selective processes, namely, [4 + 2] cycloadditions versus ene reactions. The site-specific effects of methylation on the mode selectivity and the regioselectivity of the ene reaction were studied for dienes 1e-g. A vinylogous gem effect was observed for the gamma,delta-dimethylated and alpha,gamma,delta-trimethylated substrates 1h and 1i, respectively. The corresponding phenylated substrates 1j-1 showed similar mode selectivity, as monomethylated 1j exhibited exclusively [4 + 2] reactivity while the tandem products 12 and 14 were isolated from the di- and trimethylated substrates 1k and 1l, respectively. The vinylogous gem effect favors the formation of 1,3-dienes from the substrates, and thus, secondary singlet oxygen addition was observed to give hydroperoxy-1,2-dioxenes 19 and 20 in an ene-diene transmissive cycloaddition sequence. These products were reduced to give alcohols (16, 17, and 18) or furans (24 and 25), respectively, or treated with titanium(IV) alkoxides to give the epoxy alcohols 26 and 27. The vinylogous gem effect is rationalized by DFT calculations showing that biradicals are the low-energy intermediates and that no reaction path bifurcations compete

Ene–Diene Transmissive Cycloaddition Reactions with Singlet Oxygen: The <i>Vinylogous Gem Effect</i> and Its Use for Polyoxyfunctionalization of Dienes

The singlet oxygen reactivities and regioselectivities of the model compounds <b>1b</b>–<b>d</b> were compared with those of the geminal (gem) selectivity model ethyl tiglate (<b>1a</b>). The kinetic cis effect is <i>k</i>_<i>E</i>/<i>k</i>_<i>Z</i> = 5.2 for the tiglate/angelate system <b>1a</b>/<b>1a′</b> without a change in the high gem regioselectivity. Further conjugation to vinyl groups enabled mode-selective processes, namely, [4 + 2] cycloadditions versus ene reactions. The site-specific effects of methylation on the mode selectivity and the regioselectivity of the ene reaction were studied for dienes <b>1e</b>–<b>g</b>. A vinylogous gem effect was observed for the γ,δ-dimethylated and α,γ,δ-trimethylated substrates <b>1h</b> and <b>1i</b>, respectively. The corresponding phenylated substrates <b>1j</b>–<b>l</b> showed similar mode selectivity, as monomethylated <b>1j</b> exhibited exclusively [4 + 2] reactivity while the tandem products <b>12</b> and <b>14</b> were isolated from the di- and trimethylated substrates <b>1k</b> and <b>1l</b>, respectively. The vinylogous gem effect favors the formation of 1,3-dienes from the substrates, and thus, secondary singlet oxygen addition was observed to give hydroperoxy-1,2-dioxenes <b>19</b> and <b>20</b> in an ene–diene transmissive cycloaddition sequence. These products were reduced to give alcohols (<b>16</b>, <b>17</b>, and <b>18</b>) or furans (<b>24</b> and <b>25</b>), respectively, or treated with titanium­(IV) alkoxides to give the epoxy alcohols <b>26</b> and <b>27</b>. The vinylogous gem effect is rationalized by DFT calculations showing that biradicals are the low-energy intermediates and that no reaction path bifurcations compete

Ene–Diene Transmissive Cycloaddition Reactions with Singlet Oxygen: The <i>Vinylogous Gem Effect</i> and Its Use for Polyoxyfunctionalization of Dienes

The singlet oxygen reactivities and regioselectivities of the model compounds <b>1b</b>–<b>d</b> were compared with those of the geminal (gem) selectivity model ethyl tiglate (<b>1a</b>). The kinetic cis effect is <i>k</i>_<i>E</i>/<i>k</i>_<i>Z</i> = 5.2 for the tiglate/angelate system <b>1a</b>/<b>1a′</b> without a change in the high gem regioselectivity. Further conjugation to vinyl groups enabled mode-selective processes, namely, [4 + 2] cycloadditions versus ene reactions. The site-specific effects of methylation on the mode selectivity and the regioselectivity of the ene reaction were studied for dienes <b>1e</b>–<b>g</b>. A vinylogous gem effect was observed for the γ,δ-dimethylated and α,γ,δ-trimethylated substrates <b>1h</b> and <b>1i</b>, respectively. The corresponding phenylated substrates <b>1j</b>–<b>l</b> showed similar mode selectivity, as monomethylated <b>1j</b> exhibited exclusively [4 + 2] reactivity while the tandem products <b>12</b> and <b>14</b> were isolated from the di- and trimethylated substrates <b>1k</b> and <b>1l</b>, respectively. The vinylogous gem effect favors the formation of 1,3-dienes from the substrates, and thus, secondary singlet oxygen addition was observed to give hydroperoxy-1,2-dioxenes <b>19</b> and <b>20</b> in an ene–diene transmissive cycloaddition sequence. These products were reduced to give alcohols (<b>16</b>, <b>17</b>, and <b>18</b>) or furans (<b>24</b> and <b>25</b>), respectively, or treated with titanium­(IV) alkoxides to give the epoxy alcohols <b>26</b> and <b>27</b>. The vinylogous gem effect is rationalized by DFT calculations showing that biradicals are the low-energy intermediates and that no reaction path bifurcations compete