In vitro evaluation of degradable electrospun polylactic acid/bioactive calcium phosphate ormoglass scaffolds

Nowadays, the main limitation for clinical application of scaffolds is considered to be an insufficient vascularization of the implanted platforms and healing tissues. In our studies, we proposed a novel PLA-based hybrid platform with aligned and random fibrous internal structure and incorporated calcium phosphate (CaP) ormoglass nanoparticles (0, 10, 20 and 30 wt%) as an off-the-shelf method for obtaining scaffolds with pro-angiogenic properties. Complex morphological and physicochemical evaluation of PLA-CaP ormoglass composites was performed before and after in vitro degradation test in SBF solution to assess their biological potential. The degradation process of PLA-CaP ormoglass composites was accompanied by numerous CaP-based precipitations with extended topography and cauliflower-like shape which may enhance bonding of the material with the bone tissue and accelerate the regenerative process. Random fiber orientation was preferable for CaP compounds deposition upon in vitro degradation. CaP compounds precipitated firstly for randomly oriented composite nonwovens with 20 and 30 wt% addition of ormoglass. Moreover, the preliminary bioactivity test has shown that BSA adsorbed to PLA-CaP ormoglass composites (both aligned and randomly oriented) with 20 and 30 wt% of ormoglass nanoparticles which was not observed for pure PLA scaffolds

Fast-degrading PLA/ORMOGLASS fibrous composite scaffold leads to a calcium-rich angiogenic environment

The success of scaffold implantation in acellular tissue engineering approaches relies on the ability of the material to interact properly with the biological environment. This behavior mainly depends on the design of the graft surface and, more precisely, on its capacity to biodegrade in a well-defined manner (nature of ions released, surface-to-volume ratio, dissolution profile of this release, rate of material resorption, and preservation of mechanical properties). The assessment of the biological behavior of temporary templates is therefore very important in tissue engineering, especially for composites, which usually exhibit complicated degradation behavior. Here, blended polylactic acid (PLA) calcium phosphate ORMOGLASS (organically modified glass) nanofibrous mats have been incubated up to 4 weeks in physiological simulated conditions, and their morphological, topographical, and chemical changes have been investigated. The results showed that a significant loss of inorganic phase occurred at the beginning of the immersion and the ORMOGLASS maintained a stable composition afterward throughout the degradation period. As a whole, the nanostructured scaffolds underwent fast and heterogeneous degradation. This study reveals that an angiogenic calcium-rich environment can be achieved through fast-degrading ORMOGLASS/PLA blended fibers, which seems to be an excellent alternative for guided bone regeneration

A novel hybrid nanofibrous strategy to target progenitor cells for cost-effective in situ angiogenesis

Although the impact of composites based on Ti-doped calcium phosphate glasses is low compared with that of bioglass, they have been already shown to possess great potential for bone tissue engineering. Composites made of polylactic acid (PLA) and a microparticle glass of 5TiO(2)-44.5CaO-44.5P(2)O(5)-6Na(2)O (G5) molar ratio have already demonstrated in situ osteo-and angiogenesis-triggering abilities. As many of the hybrid materials currently developed usually promote osteogenesis but still lack the ability to induce vascularization, a G5/PLA combination is a cost-effective option for obtaining new instructive scaffolds. In this study, nanostructured PLA-ORMOGLASS (organically modified glass) fibers were produced by electro-spinning, in order to fabricate extra-cellular matrix (ECM)-like substrates that simultaneously promote bone formation and vascularization. Physical-chemical and surface characterization and tensile tests demonstrated that the obtained scaffolds exhibited homogeneous morphology, higher hydrophilicity and enhanced mechanical properties than pure PLA. In vitro assays with rat mesenchymal stem cells (rMSCs) and rat endothelial progenitor cells (rEPCs) also showed that rMSCs attached and proliferated on the materials influenced by the calcium content in the environment. In vivo assays showed that hybrid composite PLA-ORMOGLASS fibers were able to promote the formation of blood vessels. Thus, these novel fibers are a valid option for the design of functional materials for tissue engineering applications

Modularity and hormone sensitivity of the Drosophila melanogaster insulin receptor/target of rapamycin interaction proteome

First systematic analysis of the evolutionary conserved InR/TOR pathway interaction proteome in Drosophila.Quantitative mass spectrometry revealed that 22% of identified protein interactions are regulated by the growth hormone insulin affecting membrane proximal as well as intracellular signaling complexes.Systematic RNA interference linked a significant fraction of network components to the control of dTOR kinase activity.Combined biochemical and genetic data suggest dTTT, a dTOR-containing complex required for cell growth control by dTORC1 and dTORC2 in vivo

Humanization and Characterization of an Anti-Human TNF-α Murine Monoclonal Antibody

A murine monoclonal antibody, m357, showing the highly neutralizing activities for human tumor necrosis factor (TNF-α) was chosen to be humanized by a variable domain resurfacing approach. The non-conserved surface residues in the framework regions of both the heavy and light chain variable regions were identified via a molecular modeling of m357 built by computer-assisted homology modeling. By replacing these critical surface residues with the human counterparts, a humanized version, h357, was generated. The humanized h357 IgG1 was then stably expressed in a mammalian cell line and the purified antibody maintained the high antigen binding affinity as compared with the parental m357 based on a soluble TNF-α neutralization bioassay. Furthermore, h357 IgG1 possesses the ability to mediate antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity upon binding to cells bearing the transmembrane form of TNF-α. In a mouse model of collagen antibody-induced arthritis, h357 IgG significantly inhibited disease progression by intra-peritoneal injection of 50 µg/mouse once-daily for 9 consecutive days. These results provided a basis for the development of h357 IgG as therapeutic use

Single Cycle Structure-Based Humanization of an Anti-Nerve Growth Factor Therapeutic Antibody

Most forms of chronic pain are inadequately treated by present therapeutic options. Compelling evidence has accumulated, demonstrating that Nerve Growth Factor (NGF) is a key modulator of inflammatory and nociceptive responses, and is a promising target for the treatment of human pathologies linked to chronic and inflammatory pain. There is therefore a growing interest in the development of therapeutic molecules antagonising the NGF pathway and its nociceptor sensitization actions, among which function-blocking anti-NGF antibodies are particularly relevant candidates

Absence of polysialylated NCAM is an unfavorable prognostic phenotype for advanced stage neuroblastoma

<p>Abstract</p> <p>Background</p> <p>The expression of a neural crest stem cell marker, polysialic acid (polySia), and its main carrier, neural cell adhesion molecule (NCAM), have been detected in some malignant tumors with high metastatic activity and unfavorable prognosis, but the diagnostic and prognostic value of polySia-NCAM in neuroblastoma is unclear.</p> <p>Methods</p> <p>A tumor tissue microarray (TMA) of 36 paraffin-embedded neuroblastoma samples was utilized to detect polySia-NCAM expression with a polySia-binding fluorescent fusion protein, and polySia-NCAM expression was compared with clinical stage, age, <it>MYCN </it>amplification status, histology (INPC), and proliferation index (PI).</p> <p>Results</p> <p>PolySia-NCAM-positive neuroblastoma patients had more often metastases at diagnosis, and polySia-NCAM expression associated with advanced disease (<it>P </it>= 0.047). Most interestingly, absence of polySia-NCAM-expressing tumor cells in TMA samples, however, was a strong unfavorable prognostic factor for overall survival in advanced disease (<it>P </it>= 0.0004), especially when <it>MYCN </it>was not amplified. PolySia-NCAM-expressing bone marrow metastases were easily detected in smears, aspirates and biopsies.</p> <p>Conclusion</p> <p>PolySia-NCAM appears to be a new clinically significant molecular marker in neuroblastoma, hopefully with additional value in neuroblastoma risk stratification.</p

Using simple artificial intelligence methods for predicting amyloidogenesis in antibodies

<p>Abstract</p> <p>Background</p> <p>All polypeptide backbones have the potential to form amyloid fibrils, which are associated with a number of degenerative disorders. However, the likelihood that amyloidosis would actually occur under physiological conditions depends largely on the amino acid composition of a protein. We explore using a naive Bayesian classifier and a weighted decision tree for predicting the amyloidogenicity of immunoglobulin sequences.</p> <p>Results</p> <p>The average accuracy based on leave-one-out (LOO) cross validation of a Bayesian classifier generated from 143 amyloidogenic sequences is 60.84%. This is consistent with the average accuracy of 61.15% for a holdout test set comprised of 103 AM and 28 non-amyloidogenic sequences. The LOO cross validation accuracy increases to 81.08% when the training set is augmented by the holdout test set. In comparison, the average classification accuracy for the holdout test set obtained using a decision tree is 78.64%. Non-amyloidogenic sequences are predicted with average LOO cross validation accuracies between 74.05% and 77.24% using the Bayesian classifier, depending on the training set size. The accuracy for the holdout test set was 89%. For the decision tree, the non-amyloidogenic prediction accuracy is 75.00%.</p> <p>Conclusions</p> <p>This exploratory study indicates that both classification methods may be promising in providing straightforward predictions on the amyloidogenicity of a sequence. Nevertheless, the number of available sequences that satisfy the premises of this study are limited, and are consequently smaller than the ideal training set size. Increasing the size of the training set clearly increases the accuracy, and the expansion of the training set to include not only more derivatives, but more alignments, would make the method more sound. The accuracy of the classifiers may also be improved when additional factors, such as structural and physico-chemical data, are considered. The development of this type of classifier has significant applications in evaluating engineered antibodies, and may be adapted for evaluating engineered proteins in general.</p

Specific Binding of the Pathogenic Prion Isoform: Development and Characterization of a Humanized Single-Chain Variable Antibody Fragment

Murine monoclonal antibody V5B2 which specifically recognizes the pathogenic form of the prion protein represents a potentially valuable tool in diagnostics or therapy of prion diseases. As murine antibodies elicit immune response in human, only modified forms can be used for therapeutic applications. We humanized a single-chain V5B2 antibody using variable domain resurfacing approach guided by computer modelling. Design based on sequence alignments and computer modelling resulted in a humanized version bearing 13 mutations compared to initial murine scFv. The humanized scFv was expressed in a dedicated bacterial system and purified by metal-affinity chromatography. Unaltered binding affinity to the original antigen was demonstrated by ELISA and maintained binding specificity was proved by Western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, humanized scFv specific for the pathogenic form of the prion protein might become a potential therapeutic reagent