Use of Native Lactococci as Vehicles for Delivery of DNA into Mammalian Epithelial Cells

The use of the food-grade bacterium Lactococcus lactis as a DNA delivery vehicle at the mucosal level is an attractive DNA vaccination strategy. Previous experiments showed that recombinant L. lactis expressing the Listeria monocytogenes inlA gene can deliver a functional gene into mammalian cells. Here, we explored the potential use of noninvasive L. lactis strains as a DNA delivery vehicle. We constructed two Escherichia coli-L. lactis shuttle plasmids, pLIG:BLG1 and pLIG:BLG2, containing a eukaryotic expression cassette with the cDNA of bovine β-lactoglobulin (BLG). The greatest BLG expression after transfection of Cos-7 cells was obtained with pLIG:BLG1, which was then used to transform L. lactis MG1363. The resulting L. lactis strain MG1363(pLIG:BLG1) was not able to express BLG. The potential of L. lactis as a DNA delivery vehicle was analyzed by detection of BLG in Caco-2 human colon carcinoma cells after 3 h of coincubation with (i) purified pLIG:BLG1, (ii) MG1363(pLIG:BLG1), (iii) a mix of MG1363(pLIG) and purified pLIG:BLG1, and (iv) MG1363. Both BLG cDNA and BLG expression were detected only in Caco-2 cells coincubated with MG1363(pLIG:BLG1). There was a decrease in the BLG cDNA level in Caco-2 cells between 24 and 48 h after coincubation. BLG expression by Caco-2 cells started at 24 h and increased between 24 and 72 h. BLG secretion by Caco-2 cells started 48 h after coincubation with MG1363(pLIG:BLG1). We conclude that lactococci can deliver BLG cDNA into mammalian epithelial cells, demonstrating their potential to deliver in vivo a DNA vaccine.     

Heterogeneity of mutations in the uroporphyrinogen III synthase gene in congenital erythropoietic porphyria

Summary Congenital erythropoietic porphyria (CEP) or Günther's disease is an inborn error of heme biosynthesis transmitted as an autosomal recessive trait and characterized by a profound deficiency of uroporphyrinogen III synthase (UROIIIS) activity. We have previously described two missense mutations in the UROIIIS gene, confirming that the primary defect responsible for CEP is a structural alteration of this gene. We have extended our work to 5 additional unrelated families. Two new point mutations, a deletion and an insertion have been found in the messenger RNA. Our study shows that a molecular heterogeneity of the mutations exists in Günther's disease. One mutation (C73R), however, appears to be more frequent than the others. Finally, the different normal and mutated proteins have been expressed in Escherichia coli to determine the consequence of the mutations on the enzyme activity.     

Complex estimates of evolutionary relationships in Tarentola mauritanica (Reptilia: Gekkonidae) derived from mitochondrial DNA sequences

Mitochondrial DNA (12S rRNA, 16S rRNA) sequences were analysed within Tarentola mauritanica and other selected species of Tarentola. Several highly genetically distinct lineages occur in North Africa, revealing phylogroups in southern and central Morocco, northern Morocco, Algeria, Tunisia, and Libya. A single haplotype characterizes populations across Spain, Portugal, Italy, Menorca, Crete, and Tunisia raising the possibility of an anthropogenic introduction followed by rapid population expansion throughout southern Europe. T. mauritanica is paraphyletic with respect to T. angustimentalis, a Canary islands endemic. The high genetic diversity observed across North Africa suggests T. mauritanica may represent a species complex.     

The G/C915 polymorphism of transforming growth factor beta1 is associated with human longevity: a study in Italian centenarians

Sequence variations in a variety of pro- or anti-inflammatory cytokine genes have been found to influence successful aging and longevity. Because of the role played by the transforming growth factor beta1 (TGF-beta1) cytokine in inflammation and regulation of immune responses, the variability of the TGF-beta1 gene may affect longevity by playing a role in inflamm-aging. Two polymorphisms, G/A -800 and C/T -509, located in the 5' region, and two missense polymorphisms, T/C 869 and G/C 915 which change (Leu > Pro)10 and (Arg > Pro)25, respectively, located in the signal peptide, were analysed in 419 subjects from Northern and Central Italy, including 172 centenarians and 247 younger controls. In addition, the effects of the TGF-beta1 genetic variability on plasma levels of the biologically active form (naturally processed) of this cytokine were studied in 143 randomly selected subjects, including 73 centenarians. Significant differences were found at the +915 site as far as the C allele and GC genotype were concerned, both of them being lower in centenarians than in young controls (P=0.034 and 0.028, respectively), but none of the other tested genetic variants was significantly different between centenarians and controls. Moreover, a particular haplotype combination (G -800/C -509/C 869/C 915) was notably lower in centenarians than in younger individuals (P=0.007). Finally, active TGF-beta1 plasma levels were significantly increased in the elderly group, but no relationship with TGF-beta1 genotypes was observed. These results suggest that, at least in this population, the variability of the TGF-beta1 gene influences longevity and that the age-related increase in plasma levels of active TGF-beta1 seems not to be genetically regulated.     

Evolution of Collective Behaviors for a Real Swarm of Aquatic Surface Robots

Swarm robotics is a promising approach for the coordination of large numbers of robots. While previous studies have shown that evolutionary robotics techniques can be applied to obtain robust and efficient self-organized behaviors for robot swarms, most studies have been conducted in simulation, and the few that have been conducted on real robots have been confined to laboratory environments. In this paper, we demonstrate for the first time a swarm robotics system with evolved control successfully operating in a real and uncontrolled environment. We evolve neural network-based controllers in simulation for canonical swarm robotics tasks, namely homing, dispersion, clustering, and monitoring. We then assess the performance of the controllers on a real swarm of up to ten aquatic surface robots. Our results show that the evolved controllers transfer successfully to real robots and achieve a performance similar to the performance obtained in simulation. We validate that the evolved controllers display key properties of swarm intelligence-based control, namely scalability, flexibility, and robustness on the real swarm. We conclude with a proof-of-concept experiment in which the swarm performs a complete environmental monitoring task by combining multiple evolved controllers.     

Mutations and genomic islands can explain the strain dependency of sugar utilization in 21 strains of Propionibacterium freudenreichii

Background

Propionibacterium freudenreichii (PF) is an actinobacterium used in cheese technology and for its probiotic properties. PF is also extremely adaptable to several ecological niches and can grow on a variety of carbon and nitrogen sources. The aim of this work was to discover the genetic basis for strain-dependent traits related to its ability to use specific carbon sources. High-throughput sequencing technologies were ideal for this purpose as they have the potential to decipher genomic diversity at a moderate cost.

Results

21 strains of PF were sequenced and the genomes were assembled de novo. Scaffolds were ordered by comparison with the complete reference genome CIRM-BIA1, obtained previously using traditional Sanger sequencing. Automatic functional annotation and manual curation were performed. Each gene was attributed to either the core genome or an accessory genome. The ability of the 21 strains to degrade 50 different sugars was evaluated. Thirty-three sugars were degraded by none of the sequenced strains whereas eight sugars were degraded by all of them. The corresponding genes were present in the core genome. Lactose, melibiose and xylitol were only used by some strains. In this case, the presence/absence of genes responsible for carbon uptake and degradation correlated well with the phenotypes, with the exception of xylitol. Furthermore, the simultaneous presence of these genes was in line the metabolic pathways described previously in other species. We also considered the genetic origin (transduction, rearrangement) of the corresponding genomic islands. Ribose and gluconate were degraded to a greater or lesser extent (quantitative phenotype) by some strains. For these sugars, the phenotypes could not be explained by the presence/absence of a gene but correlated with the premature appearance of a stop codon interrupting protein synthesis and preventing the catabolism of corresponding carbon sources.

Conclusion

These results illustrate (i) the power of correlation studies to discover the genetic basis of binary strain-dependent traits, and (ii) the plasticity of PF chromosomes, probably resulting from horizontal transfers, duplications, transpositions and an accumulation of mutations. Knowledge of the genetic basis of nitrogen and sugar degradation opens up new strategies for the screening of PF strain collections to enable optimum cheese starter, probiotic and white biotechnology applications.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1467-7) contains supplementary material, which is available to authorized users.     

The phage display technique: advantages and recent patents

Phage display technology has advanced considerably since its creation, and the number of research projects using this technique is constantly increasing, generating numerous antibody and antigen libraries. These libraries, besides expediting library screening, improving selection methods and allowing evaluation of novel applications, have great potential for the development of new vaccines, drugs and diagnosis tests. Consequently, patent registries for the protection of these sequences are essential.