Guillaume Vidal

Lack of Respiratory Chain Complex I Impairs Alternative Oxidase Engagement and Modulates Redox Signaling during Elicitor-Induced Cell Death in Tobacco

Alternative oxidase (AOX) functions in stress resistance by preventing accumulation of reactive oxygen species (ROS), but little is known about in vivo partitioning of electron flow between AOX and the cytochrome pathway. We investigated the relationships between AOX expression and in vivo activity in Nicotiana sylvestris and the complex I–deficient CMSII mutant in response to a cell death elicitor. While a specific AOX1 isoform in the active reduced state was constitutively overexpressed in CMSII, partitioning through the alternative pathway was similar to the wild type. Lack of correlation between AOX content and activity indicates severe metabolic constraints in nonstressed mutant leaves. The bacterial elicitor harpin NEa induced similar timing and extent of cell death and a twofold respiratory burst in both genotypes with little change in AOX amounts. However, partitioning to AOX was increased twofold in the wild type but remained unchanged in CMSII. Oxidative phosphorylation modeling indicated a twofold ATP increase in both genotypes. By contrast, mitochondrial superoxide dismutase activity and reduced forms of ascorbate and glutathione were higher in CMSII than in the wild type. These results demonstrate genetically programmed flexibility of plant respiratory routes and antioxidants in response to elicitors and suggest that sustained ATP production, rather than AOX activity by itself or mitochondrial ROS, might be important for in planta cell death.

Enhanced cellular adhesion on titanium by silk functionalized with titanium binding and RGD peptides.

Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. A quartz crystal microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived arginine-glycine-aspartic acid (RGD) peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by scanning electron microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell-biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required.

Method to form a fiber/growth factor dual-gradient along electrospun silk for nerve regeneration.

Concentration gradients of guidance molecules influence cell behavior and growth in biological tissues and are therefore of interest for the design of biomedical scaffolds for regenerative medicine. We developed an electrospining method to generate a dual-gradient of bioactive molecules and fiber density along electrospun nanofibers without any post spinning treatment. Functionalization with fluorescent molecules demonstrated the efficiency of the method to generate a discontinuous concentration gradient along the aligned fibers. As a proof of concept for tissue engineering, the silk nanofibers were functionalized with increasing concentrations of nerve growth factor (NGF) and the biological activity was assessed and quantified with rat dorsal root ganglion (DRG) neurons cultures. Protein assays showed the absence of passive release of NGF from the functionalized fibers. The results demonstrated that the NGF concentration gradient led to an oriented and increased growth of DRG neurons (417.6 ± 55.7 μm) compared to a single uniform NGF concentration (264.5 ± 37.6 μm). The easy-to-use electrospinning technique combined with the multiple molecules that can be used for fiber functionalization makes this technique versatile for a broad range of applications from biosensors to regenerative medicine.

Light and Oxygen Are Not Required for Harpin-induced Cell Death

Nicotiana sylvestris leaves challenged by the bacterial elicitor harpin NEa were used as a model system in which to determine the respective roles of light, oxygen, photosynthesis, and respiration in the programmed cell death response in plants. The appearance of cell death markers, such as membrane damage, nuclear fragmentation, and induction of the stress-responsive element Tnt1, was observed in all conditions. However, the cell death process was delayed in the dark compared with the light, despite a similar accumulation of superoxide and hydrogen peroxide in the chloroplasts. In contrast, harpin-induced cell death was accelerated under very low oxygen (<0.1% O2) compared with air. Oxygen deprivation impaired accumulation of chloroplastic reactive oxygen species (ROS) and the induction of cytosolic antioxidant genes in both the light and the dark. It also attenuates the collapse of photosynthetic capacity and the respiratory burst driven by mitochondrial alternative oxidase activity observed in air. Since alternative oxidase is known to limit overreduction of the respiratory chain, these results strongly suggest that mitochondrial ROS accumulate in leaves elicited under low oxygen. We conclude that the harpin-induced cell death does not require ROS accumulation in the apoplast or in the chloroplasts but that mitochondrial ROS could be important in the orchestration of the cell suicide program.

Recent Strategies in Tissue Engineering for Guided Peripheral Nerve Regeneration.

The repair of large crushed or sectioned segments of peripheral nerves remains a challenge in regenerative medicine due to the complexity of the biological environment and the lack of proper biomaterials and architecture to foster reconstruction. Traditionally such reconstruction is only achieved by using fresh human tissue as a surrogate for the absence of the nerve. However, recent focus in the field has been on new polymer structures and specific biofunctionalization to achieve the goal of peripheral nerve regeneration by developing artificial nerve prostheses. This review presents various tested approaches as well their effectiveness for nerve regrowth and functional recovery.

Complementary Effects of Two Growth Factors in Multifunctionalized Silk Nanofibers for Nerve Reconstruction

With the aim of forming bioactive guides for peripheral nerve regeneration, silk fibroin was electrospun to obtain aligned nanofibers. These fibers were functionalized by incorporating Nerve Growth Factor (NGF) and Ciliary NeuroTrophic Factor (CNTF) during electrospinning. PC12 cells grown on the fibers confirmed the bioavailability and bioactivity of the NGF, which was not significantly released from the fibers. Primary neurons from rat dorsal root ganglia (DRGs) were grown on the nanofibers and anchored to the fibers and grew in a directional fashion based on the fiber orientation, and as confirmed by growth cone morphology. These biofunctionalized nanofibers led to a 3-fold increase in neurite length at their contact, which was likely due to the NGF. Glial cell growth, alignment and migration were stimulated by the CNTF in the functionalized nanofibers. Organotypic culture of rat fetal DRGs confirmed the complementary effect of both growth factors in multifunctionalized nanofibers, which allowed glial cell migration, alignment and parallel axonal growth in structures resembling the ‘bands of Bungner’ found in situ. Graftable multi-channel conduits based on biofunctionalized aligned silk nanofibers were developed as an organized 3D scaffold. Our bioactive silk tubes thus represent new options for a biological and biocompatible nerve guidance conduit.

Organotypic culture to assess cell adhesion, growth and alignment of different organs on silk fibroin.

Glass sheets covered with aligned electrospun silk fibroin (Bombyx mori) were compared to tissue culture‐treated Thermanox® coverslips, using an organotypic culture method. Different chick embryo organ behaviours were analysed in terms of circularity, cell growth and cell adhesion after being cultivated in contact with these two materials. The circularity (cell layer shape corresponding to the trend of the biomaterials to induce a specific directionality) depends on the organ used when in contact with silk fibroin. This biomaterial induced higher cell adhesion (kidney) or lower cell adhesion (spine) compared to Thermanox. Cell growth, represented by the cell layer area (mm2), was also drastically reduced (gonad) or increased (blood vessel) on the silk fibroin. Organotypic culture is a rapid, cost effective and relatively simple method to evaluate different parameters, allowing prescreening of morphology and cytocompatibility to select the appropriate applications for new biomaterials. In the present study we compared the morphology of different organotypic cultures on orientated silk and Thermanox as growth supports to rapidly evaluate the benefit of a silk‐based biomaterial for tissue engineering. Copyright

Silk nerve: bioactive implant for peripheral nerve regeneration

Severe peripheral nerve damage affects 400,000 people each year. There are currently no effective biomaterials for nerve repair after injury or trauma. Silk proteins belong to a class of unique, high-molecular weight proteins that have found widespread use in biomaterials and regenerative medicine. These protein characteristics have robust mechanical properties, biocompatibility and biodegradability which can be enhanced with a variety of chemical modifications (Altman et al. 2003). These modifications provide tools for the attachment of growth factors, cellbinding domains and other molecules of interest to silk (Dinis et al. 2013). To manage and stimulate the nerve regeneration, we propose to develop a new type of biofunctionalised material consisting of aligned silk nanofibres produced by the electrospinning technique (Jose et al. 2012).

Multiparametric temporal analysis of the Caco-2/TC7 demonstrated functional and differentiated monolayers as early as 14 days of culture

Reducing the differentiation period for obtaining an in vitro intestinal barrier model is required to reduce the duration and cost for drug screening assays. In this frame, the Caco-2/TC7 subclone differentiation state was investigated from day 0 (D0) to day 32 (D32). As such, the expression of 45 genes (including cell junction, cell polarization, cell functionality, drug transport and metabolism genes) was followed throughout the 32 days. In parallel, the monolayer polarization and the formation of the cellular junctions were characterized by the immuno-staining of occludin, claudin-1 and actin proteins. The cell monolayer permeability was analyzed via transepithelial electric resistance measurements and paracellular transport of Lucifer Yellow. The P-gp efflux efficiency was assessed by rhodamine 123 transport. Alkaline phosphate activity was quantified to assess the cell differentiation. Three stages of differentiation were observed using the clustering of principal component analysis of the RTqPCR data and the overall assays. From D0 to D10, cells were in a proliferation stage and under-differentiated; from D14 to D21 a stable differentiation stage was reached; from D25 to D32 the epithelium seemed to enter into a post-differentiated stage. This study demonstrates that Caco-2/TC7 cells are functional and ready for use in drug screening permeability assays from 14 days in culture when compared with conventional 21 days for Caco-2 cells. In addition, this study provides a refined set of data allowing temporal and multi scale investigations, due to the intracellular kinetics and mRNA levels that can be correlated with membrane protein kinetics and functional extracellular activities. Therefore, shorter time in culture combined with a better knowledge of the cells during the time in culture will in turn help to improve the quality and cost of Caco-2/TC7 assays for drug development.