TonB-dependent outer-membrane proteins and siderophore utilization in Pseudomonas fluorescens Pf-5

The soil bacterium Pseudomonas fluorescens Pf-5 produces two siderophores, a pyoverdine and enantio-pyochelin, and its proteome includes 45 TonB-dependent outer-membrane proteins, which commonly function in uptake of siderophores and other substrates from the environment. The 45 proteins share the conserved beta-barrel and plug domains of TonB-dependent proteins but only 18 of them have an N-terminal signaling domain characteristic of TonB-dependent transducers (TBDTs), which participate in cell-surface signaling systems. Phylogenetic analyses of the 18 TBDTs and 27 TonB-dependent receptors (TBDRs), which lack the N-terminal signaling domain, suggest a complex evolutionary history including horizontal transfer among different microbial lineages. Putative functions were assigned to certain TBDRs and TBDTs in clades including well-characterized orthologs from other Pseudomonas spp. A mutant of Pf-5 with deletions in pyoverdine and enantio-pyochelin biosynthesis genes was constructed and characterized for iron-limited growth and utilization of a spectrum of siderophores. The mutant could utilize as iron sources a large number of pyoverdines with diverse structures as well as ferric citrate, heme, and the siderophores ferrichrome, ferrioxamine B, enterobactin, and aerobactin. The diversity and complexity of the TBDTs and TBDRs with roles in iron uptake clearly indicate the importance of iron in the fitness and survival of Pf-5 in the environment.

Synthesis and characterization of the [Ru(3)O(CH(3)COO)(6)(py)(2)(BPE)Ru(bpy)(2)Cl](PF(6))(2) dimer

The polymetallic [Ru(3)O(CH(3)COO)(6)(py)(2)(BPE)Ru( bpy)(2)Cl](PF(6))(2) complex (bpy = 2,2`-bipyridine, BPE = trans- 1,2-bis(4-pyridil) ethylene and py = pyridine) was assembled by the combination of an electroactive [Ru(3)O] moiety with a [ Ru( bpy) 2( BPE) Cl] photoactive centre, and its structure was determined using positive ion electrospray (ESI-MS) and tandem mass (ESI-MS/MS) spectrometry. The [Ru(3)O(CH(3)COO)(6)(py)(2)(BPE)Ru(bpy)(2)Cl] (2+) doubly charged ion of m/z 732 was mass-selected and subject to 15 eV collision-induced dissociation, leading to a specific dissociation pattern, diagnostic of the complex structure. The electronic spectra display broad bands at 409, 491 and 692 nm ascribed to the [Ru(bpy)(2)(BPE)] charge-transfer bands and to the [Ru(3)O] internal cluster transitions. The cyclic voltammetry shows five reversible waves at - 1.07 V, 0.13 V, 1.17 V, 2.91 V and - 1.29 V (vs SHE) assigned to the [Ru(3)O](-1/0/+ 1/+ 2/+3) and to the bpy (0/-1) redox processes; also a wave is observed at 0.96 V, assigned to the Ru (+2/+ 3) pair. Despite the conjugated BPE bridge, the electrochemical and spectroelectrochemical results indicate only a weak coupling through the pi-system, and preliminary photophysical essays showed the compound decomposes under visible light irradiation.

Synthesis and characterization of trans-[Ru(NO)Cl(L)(4)](PF(6))(2) (L = isonicotinamide; 4-acetylpyridine) and related species

The trans-[RUCl(2)(L)(4)], trans-[Ru(NO)Cl (L)(4)](PF(6))(2) (L = isonicotinamide and 4-acetylpyridine) and trans-[Ru(NO)(OH)(py)(4)]Cl(2) (py = pyridine) complexes have been prepared and characterized by elemental analysis, UV-visible, infrared, and (1)H NMR spectroscopies, and cyclic voltammetry. The MLCT band energies of trans-[RUCl(2)(L)(4) increase in the order 4-acpy < isn < py. The reduction potentials of trans-[RuCl(2)(L)(4)] and trans-[Ru(NO)Cl(L)(4)](2+) increase in the order py < isn < 4-acpy. The stretching band frequency. v(NO), of the nitrosyl complexes ranges from 1913 to 1852 cm(-1) indicating a nitrosonium character for the NO ligand. Due to the large pi-acceptor ability of the equatorial ligands, the coordinated water is much more acidic in the water soluble trans-[Ru(NO)(H(2)O)(py)(4)](3+) than in trans-[Ru(NO)(H(2)O)(NH(3))(4)](3+) (C) 2009 Elsevier B.V. All rights reserved.

A comparison of thermal condition between pilot- and full-scale furnaces for studying slagging and fouling propensity in PF boilers

The Australian Coal Industry Research Laboratory (ACIRL) furnace is scaled to simulate slagging and fouling in operating boilers. This requires that the gas and target temperatures, the heat flux, and the flow pattern be the same as those in real boilers. The gas and target temperatures are maintained by insulating the wall and cooling the target respectively. The flow pattern of a small burner cannot be the same as a large furnace. However, this flow pattern is partially compensated for by placing the slagging panels in three vertical locations. The paper develops the models of radiant heat transfer from the flame to the deposits both in pilot-scale and full-scale furnaces. They are used to compare the effective radiant heat transfer of the pilot- and full-scale furnaces. The experimental data both from the pilot- and full-scale furnaces are used to verify the incident heat flux and temperature profiles in the pilot- and full-scale furnaces. The results showed that the thermal condition in the pilot-scale furnace meets the requirements for studying the slagging regarding the gas temperature and the incident heat flux, particularly for the panel #1. The gas temperature in the convective section also meets the requirement for studying the fouling.

The avirulence of the cultivated PF strain of Trypanosoma cruzi: V - the evaluation of parasitologic tests after vaccination of different animal species

After vaccination with the live PF strain of Trypanosoma cruzi, 194 blood cultures were performed in 143 mice, 9 dogs, 5 Cebus monkeys and 7 human subjects. Some of these blood cultures were simultaneously done with xenodiagnosis, subinoculation in baby mice and/or culture of viscerae. The trypanosomes isolated from the few positive cases (6,1%) were incapable of infecting baby mice were considered as cases of immunotolerance. All the other tests were negative.

The avirulence of the cultivated PF strain of Trypanosoma cruzi: VI - The effect of antilymphocytic serum in dogs

The use of antilymphocytic serum (ALS) in dogs suppressed the immune response of a vacclnated and simultaneously ALS treated animal, increased the infectivity of a virulent strain and did not induce a typical infectian-disease in 3 dogs receiving the serum and the avirulent PF strain.

The un-infectivity of the PF cultivated strain of trypanosoma cruzi to mice. An evaluation through a one year period by blood cultures and histopathology

Trypanosoma cruzi of the cultivated PF strain when injected in mice per subcutaneous route, in adequate doses, is able to induce an efficient sterile immunization in the animais (for at least one year) as determined by whole blood cultures and histopathology.

Trypanosoma cruzi: avirulence of the PF strain to Callithrix marmosets

Callithrix jacchus geoffroy marmosets (HumBol. 1812) were injected once subcutaneously with 10.000 parasites/g body weight and followed for a period of six months. The PF strain of Trypanosoma cruzi was used. Follow-up was done through blood cultures, xenodiagnosis, serological tests, and ECG. A small number of normaI animais served as control.

Vaccination of Callithrix jacchus (Linné, 1758) marmosets with the PF strain of Trypanosoma cruzi

Callithrix jacchus marmosets, vaccinated more than once with high doses of the PF strain of Trypanosoma cruzi, showed a certain degree of parasitemia related to the number of parasites injected. Thirty days after vaccination, all animals were alive and showed no apparent morbid symptoms. The relationship between the dose of injected trypanosomes and the observed parasitemias is discussed and analysed as well as the immunologic incompetence of the experimental animals used.

Food allergy: Validation of the French version of the FAQLQ-PF Quality of Life Questionnaire

Background: It has been previously shown with English speaking children that food allergy clearly affects their quality of life. The first allergy quality of life questionnaire has been validated in English in 2008, however to date no questionnaire was available in French. Objectives: To validate the French version of the Food Allergy Quality of Life Questionnaire- Parent Form (FAQLQ-PF) already existing version developed and validated in English by DunnGalvin et al. Methods: The questionnaire was translated from English to French by two independent French-speaking translators and retranslated by an independent English-speaking translator. We then recruited 30 patients between 0 and 12 years with a food allergy. Parents of these children answered the questionnaire during a clinic visit. The results obtained were then analysed and compared with the results provided by DunnGalvin's study and the Food Allergy independent Measure (FAIM). Results: 27 questionnaires were fully completed and available for analysis. Median age was 6 years with a range from 18 months to 12 years. We had a girl/boy ratio of 1:1.14. A Cronbach's a correlation index of 0.748 was found. Validity was demonstrated by significant correlations between FAQLQ-PF and the FAIM. Conclusion: The French version of the FAQLQ was validated and will permit to assess degree of Quality of Life for French-speaking children with food allergy. It will be an important tool for clinical research and will allow research collaboration between French and English speaking research teams.

Evaluation of the anti-arenaviral activity of the subtilisin kexin isozyme-1/site-1 protease inhibitor PF-429242.

The cellular protease subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in the proteolytic processing of the viral envelope glycoprotein precursor (GPC) of arenaviruses, a step strictly required for production of infectious progeny. The small molecule SKI-1/S1P inhibitor PF-429242 was shown to have anti-viral activity against Old World arenaviruses. Here we extended these studies and show that PF-429242 also inhibits GPC processing and productive infection of New World arenaviruses, making PF-429242 a broadly active anti-arenaviral drug. In combination therapy, PF-429242 potentiated the anti-viral activity of ribavirin, indicating a synergism between the two drugs. A hallmark of arenaviruses is their ability to establish persistent infection in vitro and in vivo. Notably, PF-429242 was able to efficiently and rapidly clear persistent infection by arenaviruses. Interruption of drug treatment did not result in re-emergence of infection, indicating that PF-429242 treatment leads to virus extinction.

Combinatorial Anti-arenaviral Therapy with the Small Molecule SKI-1/S1P Inhibitor PF-429242 and Ribavirin

Arenaviruses are enveloped negative strand viruses that cause acute and chronic infections. Several Arenaviruses can cause severe hemorrhagic fever in humans. In West Africa Lassa virus causes several hundred thousand infections per year, while Junin, Machupo, Guanarito, and Sabia virus have emerged in South America. So far, only one drug is licensed against arenaviruses, the nucleoside analogue Ribavirin (Rib), which is effective when given early in disease, but shows only minor therapeutic effects in late stages of the infection. Previous works demonstrated that processing of the arenavirus glycoprotein precursor (GPC) by the cellular proprotein convertase site 1 protease (S1P), also known as subtilisin-kexinisozyme 1 (SKI-1), is crucial for cell-to-cell propagation of infectionand production of infectious virus. Recently, the SKI-1/S1P inhibitor PF-429242wasshownto inhibit Old World arenavirusGPCprocessing, cell-to-cell propagation, and infectious virus production. In the present study, we assessed the activity of PF-429242 against processing of the GPCs of the genetically and structurally more distant New World arenaviruses and found potent inhibition of processing of the GPCs of Junin, Machupo, and Guanarito virus. Using the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), we studied the potency of PF-429242 in the context of acute and chronic infection. In line with published data, PF-429242 potently inhibited acute LCMV infection. PF-429242 was also highly active against chronic infection and drug treatment resulted in rapid extinction of the virus without emergence of drug-resistant variants. In a combinatorial drug approach, we found that PF-429242 potentiated the anti-viral effect of Rib in treatment of acute andchronic infection. Taken together, we showed that the SKI-1/S1P inhibitor PF-429242 is broadly active against GPC processing of all major human pathogenic arenaviruses. Apart from being potent in acute infection, the drug is remarkably active in clearing chronic infection and potentiated the anti-arenaviral activity of Rib.

Genomics of secondary metabolite production by Pseudomonas fluorescens Pf-5

The complete sequence of the 7.07 Mb genome of the biological control agent Pseudomonas fluorescens Pf-5 is now available, providing a new opportunity to advance knowledge of biological control through genomics. P. fluorescens Pf-5 is a rhizosphere bacterium that suppresses seedling emergence diseases and produces a spectrum of antibiotics toxic to plant-pathogenic fungi and oomycetes. In addition to six known secondary metabolites produced by Pf-5, three novel secondary metabolite biosynthesis gene clusters identified in the genome could also contribute to biological control. The genomic sequence provides numerous clues as to mechanisms used by the bacterium to survive in the spermosphere and rhizosphere. These features include broad catabolic and transport capabilities for utilizing seed and root exudates, an expanded collection of efflux systems for defense against environmental stress and microbial competition, and the presence of 45 outer membrane receptors that should allow for the uptake of iron from a wide array of siderophores produced by soil microorganisms. As expected for a bacterium with a large genome that lives in a rapidly changing environment, Pf-5 has an extensive collection of regulatory genes, only some of which have been characterized for their roles in regulation of secondary metabolite production or biological control. Consistent with its commensal lifestyle, Pf-5 appears to lack a number of virulence and pathogenicity factors found in plant pathogen.