Wide Screening of Phage-Displayed Libraries Identifies Immune Targets in Planta

Microbe-Associated Molecular Patterns and virulence effectors are recognized by plants as a first step to mount a defence response against potential pathogens. This recognition involves a large family of extracellular membrane receptors and other immune proteins located in different sub-cellular compartments. We have used phage-display technology to express and select for Arabidopsis proteins able to bind bacterial pathogens. To rapidly identify microbe-bound phage, we developed a monitoring method based on microarrays. This combined strategy allowed for a genome-wide screening of plant proteins involved in pathogen perception. Two phage libraries for high-throughput selection were constructed from cDNA of plants infected with Pseudomonas aeruginosa PA14, or from combined samples of the virulent isolate DC3000 of Pseudomonas syringae pv. tomato and its avirulent variant avrRpt2. These three pathosystems represent different degrees in the specificity of plant-microbe interactions. Libraries cover up to 26107 different plant transcripts that can be displayed as functional proteins on the surface of T7 bacteriophage. A number of these were selected in a bio-panning assay for binding to Pseudomonas cells. Among the selected clones we isolated the ethylene response factor ATERF-1, which was able to bind the three bacterial strains in competition assays. ATERF-1 was rapidly exported from the nucleus upon infiltration of either alive or heat-killed Pseudomonas. Moreover, aterf-1 mutants exhibited enhanced susceptibility to infection. These findings suggest that ATERF-1 contains a microbe-recognition domain with a role in plant defence. To identify other putative pathogen-binding proteins on a genome-wide scale, the copy number of selected-vs.-total clones was compared by hybridizing phage cDNAs with Arabidopsis microarrays. Microarray analysis revealed a set of 472 candidates with significant fold change. Within this set defence-related genes, including well-known targets of bacterial effectors, are over-represented. Other genes non-previously related to defence can be associated through this study with general or strain-specific recognition of Pseudomonas.

Mechanism of transport of saquinavir-loaded nanostructured lipid carriers across the intestinal barrier

[EN] The aims of this work were (i) to evaluate the potential of nanostructured lipid carriers (NLCs) as a tool to 24 enhance the oral bioavailability of poorly soluble compounds using saquinavir (SQV), a BCS class IV drug 25 and P-gp substrate as a model drug, and (ii) to study NLC transport mechanisms across the intestinal barrier. 26 Three different NLC formulations were evaluated. SQV transport across Caco-2 monolayers was enhanced up 27 to 3.5-fold by NLCs compared to SQV suspension. M cells did not enhance the transport of NLCs loaded with 28 SQV. The size and amount of surfactant in the NLCs influenced SQV's permeability, the transcytosis pathway 29 and the efflux of SQV by P-gp. An NLC of size 247 nm and 1.5% (w/v) surfactant content circumvented P-gp 30 efflux and used both caveolae- and clathrin-mediated transcytosis, in contrast to the other NLC formulations, 31 which used only caveolae-mediated transcytosis. By modifying critical physicochemical parameters of the 32 NLC formulation, we were thus able to overcome the P-gp drug efflux and alter the transcytosis mechanism 33 of the nanoparticles. These findings support the use of NLCs approaches for oral delivery of poorly 34 water-soluble P-gp substrates.

Inoculation of Triatoma Virus (Dicistroviridae: Cripavirus) elicits a non-infective immune response in mice

Background: Dicistroviridae is a new family of small, non-enveloped, +ssRNA viruses pathogenic to both beneficial arthropods and insect pests. Little is known about the dicistrovirus replication mechanism or gene function, and any knowledge on these subjects comes mainly from comparisons with mammalian viruses from the Picornaviridae family. Due to its peculiar genome organization and characteristics of the per os viral transmission route, dicistroviruses make good candidates for use as biopesticides. Triatoma virus (TrV) is a pathogen of Triatoma infestans (Hemiptera: Reduviidae), one of the main vectors of the human trypanosomiasis disease called Chagas disease. TrV was postulated as a potential control agent against Chagas' vectors. Although there is no evidence that TrV nor other dicistroviruses replicate in species outside the Insecta class, the innocuousness of these viruses in humans and animals needs to be ascertained. Methods: In this study, RT-PCR and ELISA were used to detect the infectivity of this virus in Mus musculus BALB/c mice. Results: In this study we have observed that there is no significant difference in the ratio IgG2a/IgG1 in sera from animals inoculated with TrV when compared with non-inoculated animals or mice inoculated only with non-infective TrV protein capsids. Conclusions: We conclude that, under our experimental conditions, TrV is unable to replicate inmice. This study constitutes the first test to evaluate the infectivity of a dicistrovirus in a vertebrate animal model.

Breaking the 400 ppm barrier: Physical and Social implications of the recent CO2 rise

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The Carpathian range represents a weak genetic barrier in South-East Europe

Background: In the present study we have assessed whether the Carpathian Mountains represent a genetic barrier in East Europe. Therefore, we have analyzed the mtDNA of 128 native individuals of Romania: 62 of them from the North of Romania, and 66 from South Romania. Results: We have analyzed their mtDNA variability in the context of other European and Near Eastern populations through multivariate analyses. The results show that regarding the mtDNA haplogroup and haplotype distributions the Romanian groups living outside the Carpathian range (South Romania) displayed some degree of genetic differentiation compared to those living within the Carpahian range (North Romania). Conclusion: The main differentiation between the mtDNA variability of the groups from North and South Romania can be attributed to the demographic movements from East to West (prehistoric or historic) that differently affected in these regions, suggesting that the Carpathian mountain range represents a weak genetic barrier in South-East Europe.