Growth factor-induced MAPK network topology shapes Erk response determining PC-12 cell fate

The mitogen-activated protein kinase (MAPK) network is a conserved signalling module that regulates cell fate by transducing a myriad of growth-factor signals. The ability of this network to coordinate and process a variety of inputs from different growth-factor receptors into specific biological responses is, however, still not understood. We investigated how the MAPK network brings about signal specificity in PC-12 cells, a model for neuronal differentiation. Reverse engineering by modular-response analysis uncovered topological differences in the MAPK core network dependent on whether cells were activated with epidermal or neuronal growth factor (EGF or NGF). On EGF stimulation, the network exhibited negative feedback only, whereas a positive feedback was apparent on NGF stimulation. The latter allows for bi-stable Erk activation dynamics, which were indeed observed. By rewiring these regulatory feedbacks, we were able to reverse the specific cell responses to EGF and NGF. These results show that growth factor context determines the topology of the MAPK signalling network and that the resulting dynamics govern cell fate.     

Therapeutic effectiveness of orally administered transgenic low-alkaloid tobacco expressing human interleukin-10 in a mouse model of colitis

Inflammatory bowel disease (IBD) represents a spectrum of diseases in which inflammation leads to acute and chronic gut injury. It is a growing health issue for which no cure exists. The pathogenesis is multifactorial with links to infectious and environmental events that trigger disease in genetically predisposed individuals. Treatment of the two major forms of IBD, Crohn's disease and ulcerative colitis, involves the reduction of inflammation with toxic immunosuppressive drugs or blocking of the pro-inflammatory effects of tumour necrosis factor-α (TNF-α) with antibodies. Here, we show that the oral administration of transgenic low-alkaloid tobacco expressing the contra-inflammatory cytokine human interleukin-10 (hIL-10) reduces the severity of colitis by down-regulating TNF-α expression directly at the sites of inflammation in IBD-susceptible IL-10^−/– mice. hIL-10 expressed in plants is biologically active and displays resistance to gastrointestinal degradation. Dietary supplementation with plant tissue delivering up to 9 µg of hIL-10 daily for 4 weeks was well tolerated by treated mice. Gut histology was significantly improved relative to controls ( P =  0.002), and was correlated with a decrease in small bowel TNF-α mRNA levels and an increase in IL-2 and IL-1β mRNA levels. Transgenic plants expressing IL-10 to directly attenuate TNF-α expression at sites of inflammation in the gut may become a useful new approach in the luminal therapy of IBD.     

Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord

Background

Swine is an important agricultural commodity and biomedical model. Manipulation of the pig genome provides opportunity to improve production efficiency, enhance disease resistance, and add value to swine products. Genetic engineering can also expand the utility of pigs for modeling human disease, developing clinical treatment methodologies, or donating tissues for xenotransplantation. Realizing the full potential of pig genetic engineering requires translation of the complete repertoire of genetic tools currently employed in smaller model organisms to practical use in pigs.

Results

Application of transposon and recombinase technologies for manipulation of the swine genome requires characterization of their activity in pig cells. We tested four transposon systems- Sleeping Beauty, Tol2, piggyBac, and Passport in cultured porcine cells. Transposons increased the efficiency of DNA integration up to 28-fold above background and provided for precise delivery of 1 to 15 transgenes per cell. Both Cre and Flp recombinase were functional in pig cells as measured by their ability to remove a positive-negative selection cassette from 16 independent clones and over 20 independent genomic locations. We also demonstrated a Cre-dependent genetic switch capable of eliminating an intervening positive-negative selection cassette and activating GFP expression from episomal and genome-resident transposons.

Conclusion

We have demonstrated for the first time that transposons and recombinases are capable of mobilizing DNA into and out of the porcine genome in a precise and efficient manner. This study provides the basis for developing transposon and recombinase based tools for genetic engineering of the swine genome.     

A new generation of pPRIG-based retroviral vectors

Background  

Retroviral vectors are valuable tools for gene transfer. Particularly convenient are IRES-containing retroviral vectors expressing both the protein of interest and a marker protein from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV enhancer-promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient transfection. However, pAP2 contains a limited multicloning site (MCS) and shows weak eGFP expression, which previously led us to engineer an improved version, termed pPRIG, harboring: i) the wild-type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked by T7 and SP6 sequences; and iii) a HA tag encoding sequence 5' of the MCS (pPRIG HAa/b/c).     

Potential role of rhizobia to enhance chickpea-growth and yield in low fertility-soils of Tunisia

Plant growth-promoting rhizobacteria are bacteria that improve plant growth and reduce plant pathogen damages. In this study, 100 nodule bacteria were isolated from chickpea, screened for their plant growth-promoting (PGP) traits and then characterised by PCR-RFLP of 16 S rDNA. Results showed that most of the slow-growing isolates fixed nitrogen but those exhibiting fast-growth did not. Fourteen isolates solubilized inorganic phosphorus, 16 strains produced siderophores, and 17 strains produced indole acetic acid. Co-culture experiments identified three strains having an inhibitory effect against Fusarium oxysporum, the primary pathogenic fungus for chickpea in Tunisia. Rhizobia with PGP traits were assigned to Mesorhizobium ciceri, Mesorhizobium mediterraneum, Sinorhizobium meliloti and Agrobacterium tumefaciens. We noted that PGP activities were differentially distributed between M. ciceri and M. mediterraneum. The region of Mateur in northern Tunisia, with clay–silty soil, was the origin of 53% of PGP isolates. Interestingly, we found that S. meliloti and A. tumefaciens strains did not behave as parasitic nodule-bacteria but as PGP rhizobacteria useful for chickpea nutrition and health. In fact, S. meliloti strains could solubilize phosphorus, produce siderophore and auxin. The A. tumefaciens strains could perform the previous PGP traits and inhibit pathogen growth also. Finally, one candidate strain of M. ciceri (LL10)—selected for its highest symbiotic nitrogen fixation and phosphorus solubilization—was used for field experiment. The LL10 inoculation increased grain yield more than three-fold. These finding showed the potential role of rhizobia to be used as biofertilizers and biopesticides, representing low-cost and environment-friendly inputs for sustainable agriculture.

     

Operationalising emission and toxicity modelling of pesticides in LCA: the OLCA-Pest project contribution

The International Journal of Life Cycle Assessment - Current field emission modelling and toxicity characterisation of pesticides suffer from several shortcomings like mismatches between LCI...     

Specific GFP-binding artificial proteins (αRep): a new tool for in?vitro to live cell applications

A family of artificial proteins, named αRep, based on a natural family of helical repeat was previously designed. αRep members are efficiently expressed, folded and extremely stable proteins. A large αRep library was constructed creating proteins with a randomized interaction surface. In the present study, we show that the αRep library is an efficient source of tailor-made specific proteins with direct applications in biochemistry and cell biology. From this library, we selected by phage display αRep binders with nanomolar dissociation constants against the GFP. The structures of two independent αRep binders in complex with the GFP target were solved by X-ray crystallography revealing two totally different binding modes. The affinity of the selected αReps for GFP proved sufficient for practically useful applications such as pull-down experiments. αReps are disulfide free proteins and are efficiently and functionally expressed in eukaryotic cells: GFP-specific αReps are clearly sequestrated by their cognate target protein addressed to various cell compartments. These results suggest that αRep proteins with tailor-made specificity can be selected and used in living cells to track, modulate or interfere with intracellular processes.     

Influence of manufacturing processes on cell surface properties of probiotic strain <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> Lcr35<Superscript>®</Superscript>

The influence of the industrial process on the properties of probiotics, administered as complex manufactured products, has been poorly investigated. In the present study, we comparatively assessed the cell wall characteristics of the probiotic strain Lactobacillus rhamnosus Lcr35® together with three of its commercial formulations with intestinal applications. Putative secreted and transmembrane-protein-encoding genes were initially searched in silico in the genome of L. rhamnosus Lcr35®. A total of 369 candidate genes were identified which expressions were followed using a custom Lactobacillus DNA chip. Among them, 60 or 67 genes had their expression either upregulated or downregulated in the Lcr Restituo® packet or capsule formulations, compared to the native Lcr35® strain. Moreover, our data showed that the probiotic formulations (Lcr Lenio®, Lcr restituo® capsule and packet) showed a better capacity to adhere to intestinal epithelial Caco-2 cells than the native Lcr35® strain. Microbial (MATS) tests showed that the probiotic was an electron donor and that they were more hydrophilic than the native strain. The enhanced adhesion capacity of the active pharmaceutical ingredients (APIs) to epithelial Caco-2 cells and their antipathogen effect could be due to this greater surface hydrophilic character. These findings suggest that the manufacturing process influences the protein composition and the chemical properties of the cell wall. It is therefore likely that the antipathogen effect of the formulation is modulated by the industrial process. Screening of the manufactured products’ properties would therefore represent an essential step in evaluating the effects of probiotic strains.     

Probing a label-free local bend in DNA by single molecule tethered particle motion

Being capable of characterizing DNA local bending is essential to understand thoroughly many biological processes because they involve a local bending of the double helix axis, either intrinsic to the sequence or induced by the binding of proteins. Developing a method to measure DNA bend angles that does not perturb the conformation of the DNA itself or the DNA-protein complex is a challenging task. Here, we propose a joint theory-experiment high-throughput approach to rigorously measure such bend angles using the Tethered Particle Motion (TPM) technique. By carefully modeling the TPM geometry, we propose a simple formula based on a kinked Worm-Like Chain model to extract the bend angle from TPM measurements. Using constructs made of 575 base-pair DNAs with in-phase assemblies of one to seven 6A-tracts, we find that the sequence CA₆CGG induces a bend angle of 19° ± 4°. Our method is successfully compared to more theoretically complex or experimentally invasive ones such as cyclization, NMR, FRET or AFM. We further apply our procedure to TPM measurements from the literature and demonstrate that the angles of bends induced by proteins, such as Integration Host Factor (IHF) can be reliably evaluated as well.