Assembly and functional analysis of an S/MAR based episome with the cystic fibrosis transmembrane conductance regulator gene

Improving the efficacy of gene therapy vectors is still an important goal toward the development of safe and efficient gene therapy treatments. S/MAR (scaffold/matrix attached region)-based vectors are maintained extra-chromosomally in numerous cell types, which is similar to viral-based vectors. Additionally, when established as an episome, they show a very high mitotic stability. In the present study we tested the idea that addition of an S/MAR element to a CFTR (cystic fibrosis transmembrane conductance regulator) expression vector, may allow the establishment of a CFTR episome in bronchial epithelial cells. Starting from the observation that the S/MAR vector pEPI-EGFP (enhanced green fluorescence protein) is maintained as an episome in human bronchial epithelial cells, we assembled the CFTR vector pBQ-S/MAR. This vector, transfected in bronchial epithelial cells with mutated CFTR, supported long term wt CFTR expression and activity, which in turn positively impacted on the assembly of tight junctions in polarized epithelial cells. Additionally, the recovery of intact pBQ-S/MAR, but not the parental vector lacking the S/MAR element, from transfected cells after extensive proliferation, strongly suggested that pBQ-S/MAR was established as an episome. These results add a new element, the S/MAR, that can be considered to improve the persistence and safety of gene therapy vectors for cystic fibrosis pulmonary disease

Whole exome sequencing in an Italian family with isolated maxillary canine agenesis and canine eruption anomalies

Objective: The aim of this study was the clinical and molecular characterization of a family segregating a trait consisting of a phenotype specifically involving the maxillary canines, including agenesis, impaction and ectopic eruption, characterized by incomplete penetrance and variable expressivity. Design: Clinical standardized assessment of 14 family members and a whole-exome sequencing (WES) of three affected subjects were performed. WES data analyses (sequence alignment, variant calling, annotation and prioritization) were carried out using an in-house implemented pipeline. Variant filtering retained coding and splice-site high quality private and rare variants. Variant prioritization was performed taking into account both the disruptive impact and the biological relevance of individual variants and genes. Sanger sequencing was performed to validate the variants of interest and to carry out segregation analysis. Results: Prioritization of variants “by function” allowed the identification of multiple variants contributing to the trait, including two concomitant heterozygous variants in EDARADD (c.308C>T, p.Ser103Phe) and COL5A1 (c.1588G>A, p.Gly530Ser), specifically associated with a more severe phenotype (i.e. canine agenesis). Differently, heterozygous variants in genes encoding proteins with a role in the WNT pathway were shared by subjects showing a phenotype of impacted/ectopic erupted canines. Conclusions: This study characterized the genetic contribution underlying a complex trait consisting of isolated canine anomalies in a medium-sized family, highlighting the role of WNT and EDA cell signaling pathways in tooth development

Adhesion of human gingival fibroblasts/Streptococcus mitis co-culture on the nanocomposite system Chitlac-nAg

10noComposite materials are increasingly used as dental restoration. In the field of biomaterials, infections remain the main reason of dental devices failure. Silver, in the form of nanoparticles (AgNPs), ions and salt, well known for its antimicrobial properties, is used in several medical applications in order to avoid bacterial infection. To reduce both bacterial adhesion to dental devices and cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilized AgNPs with a polyelectrolyte solution containing Chitlac. Here we analyzed the proliferative and adhesive ability of human gingival fibroblasts (HGFs) on BisGMA/TEGDMA thermosets uncoated and coated with AgNPs in a coculture model system with Streptococcus mitis. After 48 h, HGFs well adhered onto both surfaces, while S. mitis cytotoxic response was higher in the presence of AgNPs coated thermosets. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release raised up to 20 %. Moreover the presence of S. mitis reduced this release mainly when HGFs adhered to Chitlac-nAg coated thermosets. The reduced secretion of collagen type I was significant in the presence of both surfaces with the co-culture system even more when saliva is added. Integrin β1 localized closely to cell membranes onto Chitlac-nAg thermosets and PKCα translocated into nuclei. These data confirm that Chitlac-nAg have a promising utilization in the field of restorative dentistry exerting their antimicrobial activity due to AgNPs without cytotoxicity for eukaryotic cells.openopenCataldi, Amelia; Gallorini, Marialucia; Di Giulio, Mara; Guarnieri, Simone; Mariggiò, Maria Addolorata; Traini, Tonino; Di Pietro, Roberta; Cellini, Luigina; Marsich, Eleonora; Sancilio, SilviaCataldi, Amelia; Gallorini, Marialucia; Di Giulio, Mara; Guarnieri, Simone; Mariggiò, Maria Addolorata; Traini, Tonino; Di Pietro, Roberta; Cellini, Luigina; Marsich, Eleonora; Sancilio, Silvi

Mechanisms of endothelial cell dysfunction in cystic fibrosis

Although cystic fibrosis (CF) patients exhibit signs of endothelial perturbation, the functions of the cystic fibrosis conductance regulator (CFTR) in vascular endothelial cells (EC) are poorly defined. We sought to uncover biological activities of endothelial CFTR, relevant for vascular homeostasis and inflammation. We examined cells from human umbilical cords (HUVEC) and pulmonary artery isolated from non-cystic fibrosis (PAEC) and CF human lungs (CF-PAEC), under static conditions or physiological shear. CFTR activity, clearly detected in HUVEC and PAEC, was markedly reduced in CF-PAEC. CFTR blockade increased endothelial permeability to macromolecules and reduced trans‑endothelial electrical resistance (TEER). Consistent with this, CF-PAEC displayed lower TEER compared to PAEC. Under shear, CFTR blockade reduced VE-cadherin and p120 catenin membrane expression and triggered the formation of paxillin- and vinculin-enriched membrane blebs that evolved in shrinking of the cell body and disruption of cell-cell contacts. These changes were accompanied by enhanced release of microvesicles, which displayed reduced capability to stimulate proliferation in recipient EC. CFTR blockade also suppressed insulin-induced NO generation by EC, likely by inhibiting eNOS and AKT phosphorylation, whereas it enhanced IL-8 release. Remarkably, phosphodiesterase inhibitors in combination with a β2 adrenergic receptor agonist corrected functional and morphological changes triggered by CFTR dysfunction in EC. Our results uncover regulatory functions of CFTR in EC, suggesting a physiological role of CFTR in the maintenance EC homeostasis and its involvement in pathogenetic aspects of CF. Moreover, our findings open avenues for novel pharmacology to control endothelial dysfunction and its consequences in CF

Intervention Program for Brazilian Children with Language Delay

The acquisition and development of language are primary in a child’s life, especially because language is one of the main means of social interaction. Therefore, it is of great importance that good language development has been assured to children and, when necessary, good intervention at their difficulties. Currently, in the field of children’s language in Brazil, different therapeutic approaches are arguable, but the necessity of development of structured therapeutic programs is verified, elaborated with technical and scientific quality so that they may stimulate the different abilities of language, aiming at considering the specificities of each child in order to minimize the difficulties. Such intervention programs would guide speech-language pathologists to plan their therapies and provide more effects in the intervention process. The aim of this study is to elaborate a stimulation program for verbal language for children with language delay. For this reason, the stimulation program was judged by peers (experts) after it was designed. The experts verified if the strategies were coherent about (a) the stimulation target and (b) the complexity level. In conclusion, the program reached the goal, since it could give direction and enhance to speech-language pathologists in cases of difficulties in verbal language

Involvement of caspace-3 in the cleavage of terminal transferase.

To investigate the in vivo role of caspase-3 in Terminal Transferase metabolism DMSO-treated RPMI-8402, a human pre-T cell line was used. In DMSO treated samples3H-dGTP incorporation and TdT phosphorylation occurs after 4 hours of treatment. After 8 hours cells undergo TdT proteolysis in addition to its inactivation. The cleavage of TdT into 32- and 58-KDa proteolytic fragments occurred simultaneously with the activation of Caspase-3, but preceded changes associated with the apoptotic process described after 48 hours of treatment. The Caspase-3 peptide inhibitor V, used as a specific inhibitor, prevented TdT proteolysis prolonging its activity and rescued cells from apoptosis. Our experiments suggest that TdT is a nuclear substrate for Caspase-3, the main apoptotic effector protease in many cell types, and that the cleavage of TdT represents a primary step in a signal cascade leading to pre-T cell apoptosis

Effects of acute and chronic low frequency electromagnetic field exposure on PC12 cells during neuronal differentiation.

The purpose of this study was to provide information about the in vitro neuritogenesis during cell exposure to extremely low frequency electromagnetic fields (ELF-EMFs) of different intensities and durations using pheochromocytoma-derived cell line (PC12 cells) as neuronal model.Proliferative rates and neuritogenesis were tested by colorimetric assay and morphological analysis, respectively; reactive oxygen species (ROS) levels and intracellular Ca(2+) variations monitored using single cell videomicroscopy.The long-lasting ELF-EMF exposure (0.1-1.0 mT) did not appear to significantly affect the biological response (proliferation and neuritogenesis). However, during the acute ELF-EMF exposure (30 min), in undifferentiated PC12 cells, there were increased ROS levels and decreased catalase activity, that, conversely, resulted increased after chronic exposure (7 days) at 1.0 mT. Acute exposure (0.1-1.0 mT) affected the spontaneous intracellular Ca(2+) variations in undifferentiated cells, in which basal intracellular Ca(2+) resulted increased after chronic exposure. In addition acute exposure affected cell response to a depolarizing agent, while basal membrane potential was not changed.Even if further studies remain necessary to identify the ROS/intracellular Ca(2+)cross-talking pathway activated by ELF-EMF exposure, we support the hypothesis that ROS and Ca(2+) could be the cellular "primum movens" of the ELF-EMF induced effects on biological systems

Solute transport and reaction in porous electrodes at high Schmidt numbers

We present lattice Boltzmann pore-scale numerical simulations of solute transport and reaction in porous electrodes at a high Schmidt number, Sc=10^2. The three-dimensional geometry of real materials is reconstructed via X-ray computed tomography. We apply a volume-averaging upscaling procedure to characterise the microstructural terms contributing to the homogenised description of the macroscopic advection–reaction–dispersion equation. We firstly focus our analysis on its asymptotic solution, while varying the rate of reaction. The results confirm the presence of two working states of the electrodes: a reaction-limited regime, governed by advective transport, and a mass-transfer-limited regime, where dispersive mechanisms play a pivotal role. For all materials, these regimes depend on a single parameter, the product of the Damk\uf6hler number and a microstructural aspect ratio. The macroscopic dispersion is determined by the spatial correlation between solute concentration and flow velocity at the pore scale. This mechanism sustains reaction in the mass-transfer-limited regime due to the spatial rearrangement of the solute transport from low-velocity to high-velocity pores. We then compare the results of pre-asymptotic transport with a macroscopic model based on effective dispersion parameters. Interestingly, the model correctly represents the transport at short characteristic times. At longer times, high reaction rates mitigate the mechanisms of heterogeneous solute transport. In the mass-transfer-limited regime, the significant yet homogeneous dispersion can thus be modelled via an effective dispersion. Finally, we formulate guidelines for the design of porous electrodes based on the microstructural aspect ratio