Staphylococcus aureus Panton-Valentine Leukocidin Is a Very Potent Cytotoxic Factor for Human Neutrophils

The role of the pore-forming Staphylococcus aureus toxin Panton-Valentine leukocidin (PVL) in severe necrotizing diseases is debated due to conflicting data from epidemiological studies of community-associated methicillin-resistant S. aureus (CA-MRSA) infections and various murine disease-models. In this study, we used neutrophils isolated from different species to evaluate the cytotoxic effect of PVL in comparison to other staphylococcal cytolytic components. Furthermore, to study the impact of PVL we expressed it heterologously in a non-virulent staphylococcal species and examined pvl-positive and pvl-negative clinical isolates as well as the strain USA300 and its pvl-negative mutant. We demonstrate that PVL induces rapid activation and cell death in human and rabbit neutrophils, but not in murine or simian cells. By contrast, the phenol-soluble modulins (PSMs), a newly identified group of cytolytic staphylococcal components, lack species-specificity. In general, after phagocytosis of bacteria different pvl-positive and pvl-negative staphylococcal strains, expressing a variety of other virulence factors (such as surface proteins), induced cell death in neutrophils, which is most likely associated with the physiological clearing function of these cells. However, the release of PVL by staphylococcal strains caused rapid and premature cell death, which is different from the physiological (and programmed) cell death of neutrophils following phagocytosis and degradation of virulent bacteria. Taken together, our results question the value of infection-models in mice and non-human primates to elucidate the impact of PVL. Our data clearly demonstrate that PVL acts differentially on neutrophils of various species and suggests that PVL has an important cytotoxic role in human neutrophils, which has major implications for the pathogenesis of CA-MRSA infections.     

Investigations of dengue-2 susceptibility and body size among Aedes aegypti populations

The mosquito Aedes aegypti (L.) (Diptera: Culicidae) is the primary global vector for dengue virus (DENV), yet considerable genetic variation exists among populations in terms of its competence to vector DENV. Variability in adult body size has also been observed among various mosquito populations and several studies have reported a relationship between body size and arbovirus dissemination, although most of these relied on artificially derived variation in body size. Here we examine the relationship between body size and disseminated DENV infection among 10 Ae. aegypti populations reared under optimum laboratory conditions. Body size variability was inferred from wing length measurements and DENV competence was evaluated as the proportion of individuals with disseminated infections following exposure to the dengue-2 JAM1409 strain. There were significant differences in mean wing lengths among populations (anova, F(9,22)= 7.10, P < 0.0001), ranging from 2.16 mm (Bangkok population) to 2.79 mm (MOYO-S [susceptible] population). We also observed significant differences among some populations in mean DENV infection rates (Waller-Duncan K-ratio t-test), ranging from 19.54% (MOYO-R [refractory] population) to 56.60% (MOYO-S population). However, we did not observe evidence for significant interactions between body size and DENV dissemination. We suggest that either the two traits are genetically independent or that our ability to detect interactions between them was limited by their respective inheritances as quantitative traits.     

Genetic Structure and Internal Rearrangements of Stable Merodiploids from BACILLUS SUBTILIS Strains Carrying the trpE26 Mutation

Transformation and transduction to tryptophan independence of strains of Bacillus subtilis carrying the "trpE26" chromosomal aberrations (a translocation and an inversion) with a "normal" 168 type strain as donor induce a tandem duplication of the thrA-ilvA region of the chromosome. The clones possessing this unstable duplication segregate besides the Trp^- some stable Trp⁺ cells which retain only part of the duplication (the trpE-ilvA region) in nontandem configuration. Such clones may also be produced directly during the crosses. The genetic map of these clones (designated as class I stable merodiploids) was constructed: they possess the tranlocation and the inversion of the trpE26 parental strain. Another type of stable Trp⁺ clones (class II) also appears, although more rarely, in similar crosses. Studies on their genetic structure revealed that they are haploid for the trpE-ilvA region and carry a nontandem duplication of the thrA-trpE region. In these clones the cysB-tre region has the orientation of the 168 type strain. The duplications in both classes are stable, that of class I being more stable than that of class II where loss of one copy of the thrA-trpE region leads to about 1% haploid cells. Detailed genetic studies on heterozygous clones from both classes have shown exchange of alleles between copies of the nontandem duplications. Models are proposed for the formation of each class of merodiploids and for recombination events taking place in them. These models imply recombination at sequences of intrachromosomal homology and (or) introduction of heterologous juncions ("novel joints") by transformation or transduction.     

Specific Interaction of the Unmodified Bacteriocin Lactococcin 972 with the Cell Wall Precursor Lipid II

Lactococcin 972 (Lcn972) is a nonlantibiotic bacteriocin that inhibits septum biosynthesis in Lactococcus lactis rather than forming pores in the cytoplasmic membrane. In this study, a deeper analysis of the molecular basis of the mode of action of Lcn972 was performed. Of several lipid cell wall precursors, only lipid II antagonized Lcn972 inhibitory activity in vivo. Likewise, Lcn972 only coprecipitated with lipid II micelles. This bacteriocin inhibited the in vitro polymerization of lipid II by the recombinant S. aureus PBP2 and the addition to lipid II of the first glycine catalyzed by FemX. These experiments demonstrate that Lcn972 specifically interacts with lipid II, the substrate of both enzymes. In the presence of Lcn972, nisin pore formation was partially hindered in whole cells. However, binding of Lcn972 to lipid II could not compete with nisin in lipid II-doped 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, possibly indicating a distinct binding site. The existence of a putative cotarget for Lcn972 activity is discussed in the context of its narrow inhibitory spectrum and the localized action at the division septum. To our knowledge, this is the first unmodified bacteriocin that binds to the cell wall precursor lipid II.     

PETIR-001, a dual inhibitor of dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN), ameliorates experimental autoimmune encephalomyelitis in SJL/J mice

Cellular dipeptidyl peptidase IV (DP IV, CD26) and amino-peptidase N (APN, CD13) play regulatory roles in T cell activation and represent potential targets for treatment of inflammatory disorders. We have developed a novel therapeutic strategy, 'peptidase-targeted Immunoregulation' (PETIR?), which simultaneously targets both cellular DP IV and APN via selective binding sites different from the active sites with a single inhibitor. To prove the therapeutic concept of PETIR? in autoimmunity of the central nervous system (CNS), we evaluated the effect of a single substance, PETIR-001, in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. Administration of PETIR-001 significantly delayed and decreased clinical signs of active EAE, when given in a therapeutic manner intraperitoneally from day 15 to day 24 after induction of EAE. Both the acute phase and the first relapse of EAE were markedly inhibited. Importantly, a similar therapeutic benefit was obtained after oral administration of PETIR-001 from day 12 to day 21 after disease induction. Our results demonstrate that PETIR-001 exhibits a therapeutic effect on EAE in SJL/J mice. Thus, PETIR? represents a novel and efficient therapeutic approach for immunotherapy of CNS inflammation.     

Functional coupling between 'R-type' Ca2+ channels and insulin secretion in the insulinoma cell line INS-1.

Among voltage-gated Ca2+ channels the non-dihydropyridine-sensitive alpha1E subunit is functionally less well characterized than the structurally related alpha1A (omega-agatoxin-IVA sensitive, P- /Q-type) and alpha1B (omega-conotoxin-GVIA sensitive, N-type) subunits. In the rat insulinoma cell line, INS-1, a tissue-specific splice variant of alpha1E (alpha1Ee) has been characterized at the mRNA and protein levels, suggesting that INS-1 cells are a suitable model for investigating the function of alpha1Ee. In alpha1E-transfected human embryonic kidney (HEK-293) cells the alpha1E-selective peptide antagonist SNX-482 (100 nM) reduces alpha1Ed- and alpha1Ee-induced Ba2+ inward currents in the absence and presence of the auxiliary subunits beta3 and alpha2delta-2 by more than 80%. The inhibition is fast and only partially reversible. No effect of SNX-482 was detected on the recombinant T-type Ca2+ channel subunits alpha1G, alpha1H, and alpha1I showing that the toxin from the venom of Hysterocrates gigas is useful as an alpha1E-selective antagonist. After blocking known components of Ca2+ channel inward current in INS-1 cells by 2 microM (+/-)-isradipine plus 0.5 microM omega-conotoxin-MVIIC, the remaining current is reduced by 100 nM SNX-482 from -12.4 +/- 1.2 pA/pF to -7.6 +/- 0.5 pA/pF (n = 9). Furthermore, in INS-1 cells, glucose- and KCl-induced insulin release are reduced by SNX-482 in a dose-dependent manner leading to the conclusion that alpha1E, in addition to L-type and non-L-type (alpha1A-mediated) Ca2+ currents, is involved in Ca2+ dependent insulin secretion of INS-1 cells.     

Revisiting Street Intersections Using Slot-Based Systems

Since their appearance at the end of the 19th century, traffic lights have been the primary mode of granting access to road intersections. Today, this centuries-old technology is challenged by advances in intelligent transportation, which are opening the way to new solutions built upon slot-based systems similar to those commonly used in aerial traffic: what we call Slot-based Intersections (SIs). Despite simulation-based evidence of the potential benefits of SIs, a comprehensive, analytical framework to compare their relative performance with traffic lights is still lacking. Here, we develop such a framework. We approach the problem in a novel way, by generalizing classical queuing theory. Having defined safety conditions, we characterize capacity and delay of SIs. In the 2-road crossing configuration, we provide a capacity-optimal SI management system. For arbitrary intersection configurations, near-optimal solutions are developed. Results theoretically show that transitioning from a traffic light system to SI has the potential of doubling capacity and significantly reducing delays. This suggests a reduction of non-linear dynamics induced by intersection bottlenecks, with positive impact on the road network. Such findings can provide transportation engineers and planners with crucial insights as they prepare to manage the transition towards a more intelligent transportation infrastructure in cities.     

Somatic cytokinesis and pollen maturation in Arabidopsis depend on TPLATE, which has domains similar to coat proteins

TPLATE was previously identified as a potential cytokinesis protein targeted to the cell plate. Disruption of TPLATE in Arabidopsis thaliana leads to the production of shriveled pollen unable to germinate. Vesicular compartmentalization of the mature pollen is dramatically altered, and large callose deposits accumulate near the intine cell wall layer. Green fluorescent protein (GFP)-tagged TPLATE expression under the control of the pollen promoter Lat52 complements the phenotype. Downregulation of TPLATE in Arabidopsis seedlings and tobacco (Nicotiana tabacum) BY-2 suspension cells results in crooked cell walls and cell plates that fail to insert into the mother wall. Besides accumulating at the cell plate, GFP-fused TPLATE is temporally targeted to a narrow zone at the cell cortex where the cell plate connects to the mother wall. TPLATE-GFP also localizes to subcellular structures that accumulate at the pollen tube exit site in germinating pollen. Ectopic callose depositions observed in mutant pollen also occur in RNA interference plants, suggesting that TPLATE is implicated in cell wall modification. TPLATE contains domains similar to adaptin and beta-COP coat proteins. These data suggest that TPLATE functions in vesicle-trafficking events required for site-specific cell wall modifications during pollen germination and for anchoring of the cell plate to the mother wall at the correct cortical position.     

Leaf gas exchange of trees in old-growth and young secondary forest stands in Sulawesi, Indonesia

In the tropics, old-growth forests are converted to other land cover types at a high rate and young secondary forest may gain in importance. Information on associated changes in leaf gas exchange and other leaf traits can be valuable for modelling biogeochemical fluxes under altered land-use patterns. We studied in situ photosynthetic parameters and stomatal conductance for water vapour in eight abundant tree species of young secondary forest and eight tree species of natural old-growth forest in Central Sulawesi, Indonesia. In sun leaves, the average maximal stomatal conductance (g _smax) in the secondary forest (SF) species was 2.1 times higher than in the old-growth forest (OGF) species. Species with a high g _smax reduced g _s sharply when vapour pressure deficit of the air increased, whereas species with a low g _smax were much less sensitive to air humidity. For area-based photosynthetic capacity (A _max-area), the SF species had a 2.3 times higher average than the OGF species. For both, g _smax and A _max-area the variation among species was higher in the OGF than in the SF. When all tree species (n=16) are considered, species means of specific leaf area (SLA), leaf N concentration and leaf P concentration were significantly correlated with g _smax and A _max-area. The strong correlation between A _max-area and foliar P (r ²=0.8) is remarkable as the alluvial soils in the study region are rich in nutrients. If the eight OGF species are analysed separately, the only significant correlation was observed between SLA and mass-based A _max; in the SF species strong correlations were found between leaf size and A _max-area and g _smax. These results show that the conversion of old-growth forest to young secondary forest in Sulawesi significantly alters tree leaf gas exchange characteristics and that chemical and structural leaf traits can be used for the prediction of these changes. The best correlations between leaf gas exchange parameters and leaf traits were obtained by different traits in the SF species, the OGF species and the entire pool of studied species.