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It is established that the human pathogen Legionella pneumophila becomes significantly augmented for infection of macrophages after intracellular growth in amoebae when compared to like-strains cultivated in laboratory media. Based on this observation, we reasoned that the most critical virulence determinants of L.p. are expressed by responding to stimuli generated by the protozoan host specifically; a process we term “protozoan-priming.” We sought to identify L.p. virulence factors that were required for replication in amoebae in order to highlight the genes necessary for production of the most infectious form of the bacterium. Using a transposon mutagenesis screen, we successfully identified 12 insertions that produced bacteria severely attenuated for growth in amoebae, while retaining a functional Dot/Icm type IVb secretion system. Seven of these insertion mutants were found dispensable for growth in macrophages, revealing attractive therapeutic targets that reside upstream of the pathogen-human interface. Two candidates identified, lpg0730 and lpg0122 were required for survival and replication in amoebae and macrophage host cells. Both genes are conserved among numerous important human pathogenic bacteria that can persist or replicate in amoebae. Each gene encodes a component of an ATP binding cassette (ABC) transport complex of unknown function. We demonstrate the lpg0730 ortholog in Francisella tularensis subsp. novicida to be essential for colonization of both protozoan and mammalian host cells, highlighting conserved survival mechanisms employed by bacteria that utilize protozoa as an environmental reservoir for replication.

The alternative sigma factor σE functions to maintain bacterial homeostasis and membrane integrity in response to extracytoplasmic stress by regulating thousands of genes both directly and indirectly. The transcriptional regulatory network governed by σE in Salmonella and E. coli has been examined using microarray, however a genome-wide analysis of σE-binding sites in Salmonella has not yet been reported. We infected macrophages with Salmonella Typhimurium over a select time course. Using chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq), 31 σE-binding sites were identified. Seventeen sites were new, which included outer membrane proteins, a quorum-sensing protein, a cell division factor, and a signal transduction modulator. The consensus sequence identified for σE in vivo binding was similar to the one previously reported, except for a conserved G and A between the -35 and -10 regions. One third of the σE-binding sites did not contain the consensus sequence, suggesting there may be alternative mechanisms by which σE modulates transcription. By dissecting direct and indirect modes of σE-mediated regulation, we found that σE activates gene expression through recognition of both canonical and reversed consensus sequence. New σE regulated genes (greA, luxS, ompA and ompX) are shown to be involved in heat shock and oxidative stress responses.

A cost-effective and efficacious influenza vaccine for use in commercial poultry farms would help protect against avian influenza outbreaks. Current influenza vaccines for poultry are expensive and subtype specific, and therefore there is an urgent need to develop a universal avian influenza vaccine. We have constructed a live bacterial vaccine against avian influenza by expressing a conserved peptide from the ectodomain of M2 antigen (M2e) on the surface of Lactococcus lactis (LL). Chickens were vaccinated intranasally with the lactococcal vaccine (LL-M2e) or subcutaneously with keyhole-limpet-hemocyanin conjugated M2e (KLH-M2e). Vaccinated and nonvaccinated birds were challenged with high pathogenic avian influenza virus A subtype H5N2. Birds vaccinated with LL-M2e or KLH-M2e had median survival times of 5.5 and 6.0 days, respectively, which were significantly longer than non-vaccinated birds (3.5 days). Birds vaccinated subcutaneously with KLH-M2e had a lower mean viral burden than either of the other two groups. However, there was a significant correlation between the time of survival and M2e-specific serum IgG. The results of these trials show that birds in both vaccinated groups had significantly (P

It is established that the human pathogen Legionella pneumophila becomes significantly augmented for infection of macrophages after intracellular growth in amoebae when compared to like-strains cultivated in laboratory media. Based on this observation, we reasoned that the most critical virulence determinants of L.p. are expressed by responding to stimuli generated by the protozoan host specifically; a process we term "protozoan-priming." We sought to identify L.p. virulence factors that were required for replication in amoebae in order to highlight the genes necessary for production of the most infectious form of the bacterium. Using a transposon mutagenesis screen, we successfully identified 12 insertions that produced bacteria severely attenuated for growth in amoebae, while retaining a functional Dot/Icmtype IVb secretion system. Seven of these insertion mutants were found dispensable for growth in macrophages, revealing attractive therapeutic targets that reside upstream of the pathogen-human interface. Two candidates identified, lpg0730 and lpg0122 were required for survival and replication in amoebae and macrophage host cells. Both genes are conserved among numerous important human pathogenic bacteria that can persist or replicate in amoebae. Each gene encodes a component of an ATP binding cassette (ABC) transport complex of unknown function. We demonstrate the lpg0730 ortholog in Francisella tularensis subsp. novicida to be essential for colonization of both protozoan and mammalian host cells, highlighting conserved survival mechanisms employed by bacteria that utilize protozoa as an environmental reservoir for replication. [ABSTRACT FROM AUTHOR]

The alternative sigma factor σE functions to maintain bacterial homeostasis and membrane integrity in response to extracytoplasmic stress by regulating thousands of genes both directly and indirectly. The transcriptional regulatory network governed by σE in Salmonella and E. coli has been examined using microarray, however a genome-wide analysis of σE–binding sites in Salmonella has not yet been reported. We infected macrophages with Salmonella Typhimurium over a select time course. Using chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq), 31 σE–binding sites were identified. Seventeen sites were new, which included outer membrane proteins, a quorum-sensing protein, a cell division factor, and a signal transduction modulator. The consensus sequence identified for σE in vivo binding was similar to the one previously reported, except for a conserved G and A between the -35 and -10 regions. One third of the σE–binding sites did not contain the consensus sequence, suggesting there may be alternative mechanisms by which σE modulates transcription. By dissecting direct and indirect modes of σE-mediated regulation, we found that σE activates gene expression through recognition of both canonical and reversed consensus sequence. New σE regulated genes (greA, luxS, ompA and ompX) are shown to be involved in heat shock and oxidative stress responses. [ABSTRACT FROM AUTHOR]

The extracytoplasmic functioning sigma factor σE is known to play an essential role for Salmonella enterica serovar Typhimurium to survive and proliferate in macrophages and mice. However, its regulatory network is not well-characterized, especially during infection. Here we used microarray to identify genes regulated by σE in Salmonella grown in three conditions: a nutrient-rich condition and two others that mimic early and late intracellular infection. We found that in each condition σE regulated different sets of genes, and notably, several global regulators. When comparing nutrient-rich and infection-like conditions, large changes were observed in the expression of genes involved in Salmonella pathogenesis island (SPI)-1 type-three secretion system (TTSS), SPI-2 TTSS, protein synthesis, and stress responses. In total, the expression of 58% of Salmonella genes was affected by σE in at least one of the three conditions. An important finding is that σE up-regulates SPI-2 genes, which are essential for Salmonella intracellular survival, by up-regulating SPI-2 activator ssrB expression at the early stage of infection and down-regulating SPI-2 repressor hns expression at a later stage. Moreover, σE is capable of countering the silencing of H-NS, releasing the expression of SPI-2 genes. This connection between σE and SPI-2 genes, combined with the global regulatory effect of σE, may account for the lethality of rpoE-deficient Salmonella in murine infection. [ABSTRACT FROM AUTHOR]

A cost-effective and efficacious influenza vaccine for use in commercial poultry farms would help protect against avian influenza outbreaks. Current influenza vaccines for poultry are expensive and subtype specific, and therefore there is an urgent need to develop a universal avian influenza vaccine. We have constructed a live bacterial vaccine against avian influenza by expressing a conserved peptide from the ectodomain of M2 antigen (M2e) on the surface of Lactococcus lactis (LL). Chickens were vaccinated intranasally with the lactococcal vaccine (LL-M2e) or subcutaneously with keyhole-limpet-hemocyanin conjugated M2e (KLHM2e). Vaccinated and nonvaccinated birds were challenged with high pathogenic avian influenza virus A subtype H5N2. Birds vaccinated with LL-M2e or KLH-M2e had median survival times of 5.5 and 6.0 days, respectively, which were significantly longer than non-vaccinated birds (3.5 days). Birds vaccinated subcutaneously with KLH-M2e had a lower mean viral burden than either of the other two groups. However, there was a significant correlation between the time of survival and M2e-specific serum IgG. The results of these trials show that birds in both vaccinated groups had significantly (P < 0.05) higher median survival times than non-vaccinated birds and that this protection could be due to M2e-specific serum IgG. [ABSTRACT FROM AUTHOR]

Phosphate based glasses (PBG) are currently of huge interest in the biomaterials field and findings have shown that their durability can be increased by doping with selected metal cations. In this study various compositions in the system (50-x)Na2O-xCaO-50P2O5 glasses, 0≤x≤50, were investigated to determine their characteristic temperatures (glass transition temperature (Tg), onset and peak temperatures of crystallisation (Tx and Tp), melting point and liquidus temperature (Tm and TJ), and also the temperature dependence of viscosity and density. The glass compositions were confirmed via EDX and the amorphous nature of the glasses was confirmed via XRD. Tg increased with increasing CaO mol% from 280±0⋅5°C to 521±01°C. The temperature dependence of viscosity revealed an increase in the temperature window for extrusion (from 26±0-66°C to 42±0-31°C) and fibre drawing (from 363±1⋅0°C to 539±0⋅9°C). The density was also seen to increase from 2⋅5 to 2⋅64 gcm-3. The increases in properties were attributed to the crosslinking of Ca2+ cations between phosphate chains which strengthened the glass network. For x≤20 mol% the activation energy for viscous flow is affected by the phosphate chain length. For 30≥x≥50 mol% the crosslinking effect of Ca2+ counters the effect of decreasing chain length, resulting in increasing activation energy for viscous flow. [ABSTRACT FROM AUTHOR]

Phosphate glass fiber of the composition 20Na2O-24MgO-16CaO-40P2O5 was produced using an in-house fiber drawing rig. The interfacial properties of the phosphate glass fiber/poly(caprolactone) (PCL) system were measured using the single fiber fragmentation test (SFFT). The system was calibrated using E-glass fibers and polypropylene system. This gave an interfacial shear strength (IFSS) of 4.1 MPa, which agrees well with other published data. The IFSS for the unsized (as drawn) phosphate glass fiber/PCL system was found to be 1.75 MPa. Fibers treated with 3-aminopropyl-triethoxy silane (APS) showed an IFSS of 3.82 MPa. X-ray photoelectron spectroscopic (XPS) analysis of unsized and silane sized fibers established the presence of silane on the fiber surface. Degradation tests of the silane treated fiber/PCL samples were carried out in deionised water at 37°C and it was found that the IFSS values decreased over time. Four others silanes were also investigated but APS gave the highest IFSS values. [ABSTRACT FROM AUTHOR]

We report results from an initial small animal study designed to provide information on the biocompatibility of a novel biodegradable composite designed for craniomaxillofacial reconstruction. Rat calvarium was chosen as a clinically analogous model, which allowed comparison between experimental groups (PCL alone, PCL/phosphate glass, or PCL/bioglass implants) and control groups (empty defects or bone grafted defects). All animals recovered well from surgery and no clinical complications were observed. Histological assessment indicated a lack of inflammatory response. The amount of new bone formation at the dural aspect of the implant was statistically significantly higher in the PCL/phosphate glass group than the other experimental groups. This study confirms, in a clinically analogous model, the promise of the novel PCL/phosphate glass composite material. Work is planned toward manufacturing scale up and clinical trials. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2010. [ABSTRACT FROM AUTHOR]

Poly(caprolactone) matrix iron phosphate glass fiber-reinforced (16Na2O-24MgO-16CaO-4Fe2O3-40P2O5) composites (10% fiber by weight) were fabricated by compression molding. Tensile strength, tensile modulus, elongation at break (%), bending strength, bending modulus and impact strength of the composites were found to be 72 MPa, 2.74 GPa, 11%, 86 MPa, 3.22 GPa and 16 kJ/m2, respectively. Degradation tests of the fibers and composites were performed in aqueous medium at 37°C. Mechanical properties of the composites retained almost 90% of their original properties after 6 months of aqueous degradation. Interfacial shear strength (IFSS) of the composite was also measured by using the single-fiber fragmentation test. The IFSS of the composite system was found to be 4.01 MPa. [ABSTRACT FROM AUTHOR]

We investigate the fabrication of continuous carbon fiber/poly(ε-caprolactone) composites for biomedical applications. Two fabrication methods: in-situ polymerization and film stacking are compared at two different thicknesses. Analysis of void content produced via the two fabrication methods showed that the in-situ polymerization specimens had considerably lower void content than the film stacked samples. Significantly higher mechanical properties were obtained for the in-situ polymerized samples, and an almost three-fold increase in properties was seen as compared to the film stacked samples. Scanning electron microscopy images of surfaces supported the findings above. Dynamic mechanical analysis revealed higher dynamic modulus for the in-situ polymerized samples which was consistent with the above results. [ABSTRACT FROM AUTHOR]

The article explores the kinetics of ε-caprolactone polymerization in the presence of Sn(Oct)2/BF3 dual catalyst. Resuls have demonstrated an induction period for both neat Sn(Oct)2 catalyst and Sn(Oct)2/BF3 dual catalyst, whereas there is no induction period for neat BF3 catalyst. The length of the induction period for the Sn(Oct)2-catalyzed reactions was not affected by the presence of BF3.

Polymers prepared from polylactic acid (PLA) have found a multitude of uses as medical devices. The main advantage of having a material that degrades is so that an implant would not necessitate a second surgical event for removal. In addition, the biodegradation may offer other advantages. In this study, fibers produced from a quaternary phosphate‐based glass (PBG) in the system 50P2O5‐40CaO‐5Na2O‐5Fe2O3 (nontreated and heat‐treated) were used to reinforce the biodegradable polymer, PLA. Fiber properties were investigated, along with the mechanical and degradation properties and cytocompatibility of the composites produced. Retention of mechanical properties overtime was also evaluated. The mean fiber strength for the phosphate glass fibers was 456 MPa with a modulus value of 51.5 GPa. Weibull analysis revealed a shape and scale parameter value of 3.37 and 508, respectively. The flexural strength of the composites matched that for cortical bone; however, the modulus values were lower than those required for cortical bone. After 6 weeks of degradation in deionized water, 50% of the strength values obtained was maintained. The composite degradation properties revealed a 14% mass loss for the nontreated and a 10% mass loss for the heat‐treated fiber composites. It was also seen that by heat‐treating the fibers, chemical and physical degradation occurred much slower. The pH profiles also revealed that nontreated fibers degraded quicker, thus correlating well with the degradation profiles. The in vitro cell culture experiments revealed both PLA (alone) and the heat‐treated fiber composites maintained higher cell viability as compared to the nontreated fiber composites. This was attributed to the slower degradation release profiles of the heat‐treated composites as compared to the nontreated fiber composites. SEM analyses revealed a porous structure after degradation, and it is clear that there are possibilities here to tailor the distribution of porosity within polymer matrices. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009 [ABSTRACT FROM AUTHOR]