Bioengineering of AAV2 Capsid at Specific Serine, Threonine, or Lysine Residues Improves Its Transduction Efficiency in Vitro and in Vivo

We hypothesized that the AAV2 vector is targeted for destruction in the cytoplasm by the host cellular kinase/ubiquitination/proteasomal machinery and that modification of their targets on AAV2 capsid may improve its transduction efficiency. In vitro analysis with pharmacological inhibitors of cellular serine/threonine kinases (protein kinase A, protein kinase C, casein kinase II) showed an increase (20-90%) on AAV2-mediated gene expression. The three-dimensional structure of AAV2 capsid was then analyzed to predict the sites of ubiquitination and phosphorylation. Three phosphodegrons, which are the phosphorylation sites recognized as degradation signals by ubiquitin ligases, were identified. Mutation targets comprising eight serine (S) or seven threonine (T) or nine lysine (K) residues were selected in and around phosphodegrons on the basis of their solvent accessibility, overlap with the receptor binding regions, overlap with interaction interfaces of capsid proteins, and their evolutionary conservation across AAV serotypes. AAV2-EGFP vectors with the wild-type (WT) capsid or mutant capsids (15 S/T -> alanine A] or 9 K -> arginine R] single mutant or 2 double K -> R mutants) were then evaluated in vitro. The transduction efficiencies of 11 S/T -> A and 7 K -> R vectors were significantly higher (similar to 63-90%) than the AAV2-WT vectors (similar to 30-40%). Further, hepatic gene transfer of these mutant vectors in vivo resulted in higher vector copy numbers (up to 4.9-fold) and transgene expression (up to 14-fold) than observed from the AAV2-WT vector. One of the mutant vectors, S489A, generated similar to 8-fold fewer antibodies that could be cross-neutralized by AAV2-WT. This study thus demonstrates the feasibility of the use of these novel AAV2 capsid mutant vectors in hepatic gene therapy.

Cell Reprogramming, IPS Limitations, and Overcoming Strategies in Dental Bioengineering

8 p.

Selective biodegradation of keratin matrix in feather rachis reveals classic bioengineering

Flight necessitates that the feather rachis is extremely tough and light. Yet, the crucial filamentous hierarchy of the rachis is unknown—study hindered by the tight chemical bonding between the filaments and matrix. We used novel microbial biodegradation to delineate the fibres of the rachidial cortex in situ. It revealed the thickest keratin filaments known to date (factor >10), approximately 6 µm thick, extending predominantly axially but with a small outer circumferential component. Near-periodic thickened nodes of the fibres are staggered with those in adjacent fibres in two- and three-dimensional planes, creating a fibre–matrix texture with high attributes for crack stopping and resistance to transverse cutting. Close association of the fibre layer with the underlying ‘spongy’ medulloid pith indicates the potential for higher buckling loads and greater elastic recoil. Strikingly, the fibres are similar in dimensions and form to the free filaments of the feather vane and plumulaceous and embryonic down, the syncitial barbules, but, identified for the first time in 140+ years of study in a new location—as a major structural component of the rachis. Early in feather evolution, syncitial barbules were consolidated in a robust central rachis, definitively characterizing the avian lineage of keratin.

Biomechanics Studies in Dentistry: Bioengineering Applied in Oral Implantology

The application of engineering knowledge in dentistry has helped the understanding of biomechanics aspects related to osseointegrated implants. Several techniques have been used to evaluate the biomechanical load oil implants comprising the use of photoelastic stress analysis, finite element stress analysis, and strain-gauge analysis. Therefore, the purpose of this Study was to describe engineering methods used in dentistry to evaluate the biomechanical behavior of osseointegrated implants. Photoelasticity provides good qualitative information oil the overall location and concentration of stresses but produces limited quantitative information. The method serves as ail important tool for determining the critical stress points in a material and is often used for determining stress concentration factors in irregular geometries. The application of strain-gauge method oil dental implants is based oil the use of electrical resistance strain gauges and its associated equipment and provides both in vitro and vivo measurements strains under static and dynamic loads. However, strain-gauge method provides only the data regarding strain at the gauge. Finite element analysis can Simulate stress using a computer-created model to calculate stress, strain, and displacement. Such analysis has the advantage of allowing several conditions to be changed easily and allows measurement of stress distribution around implants at optional points that are difficult to examine clinically All the 3 methodologies call be useful to evaluate biomechanical implant behavior close to the clinical condition but the researcher should have enough knowledge in model fabrication (experimental delineation) and results analysis.

Cellular behavior as a dynamic field for exploring bone bioengineering: A closer look at cell-biomaterial interface

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Bone morphogenetic proteins: promising molecules for bone healing, bioengineering, and regenerative medicine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)