Mutations in protein kinase Cγ promote spinocerebellar ataxia type 14 by impairing kinase autoinhibition.

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline variants in the diacylglycerol (DAG)/Ca2+-regulated protein kinase Cγ (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. Most of the identified mutations cluster in the DAG-sensing C1 domains. Here, we found with a FRET-based activity reporter that SCA14-associated PKCγ mutations, including a previously undescribed variant, D115Y, enhanced the basal activity of the kinase by compromising its autoinhibition. Unlike other mutations in PKC that impair its autoinhibition but lead to its degradation, the C1 domain mutations protected PKCγ from such down-regulation. This enhanced basal signaling rewired the brain phosphoproteome, as revealed by phosphoproteomic analysis of cerebella from mice expressing a human SCA14-associated H101Y mutant PKCγ transgene. Mutations that induced a high basal activity in vitro were associated with earlier average age of onset in patients. Furthermore, the extent of disrupted autoinhibition, but not agonist-stimulated activity, correlated with disease severity. Molecular modeling indicated that almost all SCA14 variants not within the C1 domain were located at interfaces with the C1B domain, suggesting that mutations in and proximal to the C1B domain are a susceptibility for SCA14 because they uniquely enhance PKCγ basal activity while protecting the enzyme from down-regulation. These results provide insight into how PKCγ activation is modulated and how deregulation of the cerebellar phosphoproteome by SCA14-associated mutations affects disease progression.

Assessment of Elastic-Plastic Fracture Behavior of Cortical Bone Using a Small Punch Testing Technique.

The fracture properties of cortical bone are directly coupled to its complex hierarchical structure. The limited availability of bone material from many anatomic locations creates challenges for assessing the effect of bone heterogeneity and anisotropy on fracture properties. The small punch technique was employed to examine the fracture behavior of cortical bone in terms of area under the curve values obtained from load-load point displacement behavior. Fracture toughness of cortical bone was also determined in terms of J-toughness values obtained using a compact tension (CT) test. Area under the curve values obtained from the small punch test were correlated with the J-toughness values of cortical bone. The effects of bone density and compositional parameters on area under the curve and Jtoughness values were also analyzed using linear and multiple regression analysis. Area under the curve and J-toughness values are strongly and positively correlated. Bone density and %mineral content are positively correlated with both area under the curve and J-toughness values. The multiple regression analysis outcomes support these results. Overall, the findings support the hypothesis that area under the curve values obtained from small punch tests can be used to assess the fracture behavior of cortical bone.

Studies on the Stress-Strain Relationship Bovine Cortical Bone Based on Ramberg-Osgood Equation.

Bone is a complex material that exhibits an amount of plasticity before bone fracture takes place, where the nonlinear relationship between stress and strain is of importance to understand the mechanism behind the fracture. This brief presents our study on the examination of the stress-strain relationship of bovine femoral cortical bone and the relationship representation by employing the Ramberg-Osgood (R-O) equation. Samples were taken and prepared from different locations (upper, middle, and lower) of bone diaphysis and were then subjected to the uniaxial tensile tests under longitudinal and transverse loading conditions, respectively. The stress-strain curves obtained from tests were analyzed via linear regression analysis based on the R-O equation. Our results illustrated that the R-O equation is appropriate to describe the nonlinear stress-strain behavior of cortical bone, while the values of equation parameters vary with the sample locations (upper, middle, and lower) and loading conditions (longitudinal and transverse).

Modulating mechanical behaviour of 3D-printed cartilage-mimetic PCL scaffolds: influence of molecular weight and pore geometry.

Three-dimensional (3D)-printed poly(ε)-caprolactone (PCL)-based scaffolds are increasingly being explored for cartilage tissue engineering (CTE) applications. However, ensuring that the mechanical properties of these PCL-based constructs are comparable to that of articular cartilage that they are meant to regenerate is an area that has been under-explored. This paper presents the effects of PCL's molecular weight (MW) and scaffold's pore geometric configurations; strand size (SZ), strand spacing (SS), and strand orientation (SO), on mechanical properties of 3D-printed PCL scaffolds. The results illustrate that MW has significant effect on compressive moduli and yield strength of 3D-printed PCL scaffolds. Specifically, PCL with MW of 45 K was a more feasible choice for fabrication of visco-elastic, flexible and load-bearing PCL scaffolds. Furthermore, pore geometric configurations; SZ, SS, and SO, all significantly affect on tensile moduli of scaffolds. However, only SZ and SS have statistically significant effects on compressive moduli and porosity of these scaffolds. That said, inverse linear relationship was observed between porosity and mechanical properties of 3D-printed PCL scaffolds in Pearson's correlation test. Altogether, this study illustrates that modulating MW of PCL and pore geometrical configurations of the scaffolds enabled design and fabrication of PCL scaffolds with mechanical and biomimetic properties that better mimic mechanical behaviour of human articular cartilage. Thus, the modulated PCL scaffold proposed in this study is a framework that offers great potentials for CTE applications.

Characterization of Mechanical Properties of Tissue Scaffolds by Phase Contrast Imaging and Finite Element Modeling.

In tissue engineering, the cell and scaffold approach has shown promise as a treatment to regenerate diseased and/or damaged tissue. In this treatment, an artificial construct (scaffold) is seeded with cells, which organize and proliferate into new tissue. The scaffold itself biodegrades with time, leaving behind only newly formed tissue. The degradation qualities of the scaffold are critical during the treatment period, since the change in the mechanical properties of the scaffold with time can influence cell behavior. To observe in time the scaffold's mechanical properties, a straightforward method is to deform the scaffold and then characterize scaffold deflection accordingly. However, experimentally observing the scaffold deflection is challenging. This paper presents a novel study on characterization of mechanical properties of scaffolds by phase contrast imaging and finite element modeling, which specifically includes scaffold fabrication, scaffold imaging, image analysis, and finite elements (FEs) modeling of the scaffold mechanical properties. The innovation of the work rests on the use of in-line phase contrast X-ray imaging at 20 KeV to characterize tissue scaffold deformation caused by ultrasound radiation forces and the use of the Fourier transform to identify movement. Once deformation has been determined experimentally, it is then compared with the predictions given by the forward solution of a finite element model. A consideration of the number of separate loading conditions necessary to uniquely identify the material properties of transversely isotropic and fully orthotropic scaffolds is also presented, along with the use of an FE as a form of regularization.

Use of the polycation polyethyleneimine to improve the physical properties of alginate-hyaluronic acid hydrogel during fabrication of tissue repair scaffolds.

Recently alginate-based tissue repair scaffolds fabricated using 3D printing techniques have been extensively examined for use in tissue engineering applications. However, their physical and mechanical properties are unfavorable for many tissue engineering applications because these properties are poorly controlled during the fabrication process. Some improvement of alginate gel properties can be realized by addition of hyaluronic acid (HA), and this may also improve the ability of cells to interact with the gel. Here, we report improvement of the physical properties of alginate-HA gel scaffolds by the addition of the polycation polyethyleneimine (PEI) during the fabrication process in order to stabilize alginate molecular structure through the formation of a polyelectrolyte complex. We find that PEI has a significant beneficial influence on alginate-HA scaffold physical properties, including a reduction in the degree of gel swelling, a reduction in scaffold degradation rate, and an increase in the Young's modulus of the gel. Further study shows that fabrication of alginate-HA gels with PEI increases the encapsulation efficiency of bovine serum albumin, a model protein, and reduces the subsequent initial protein release rate. However, it was also found that survival of Schwann cells or ATDC-5 chondrogenic cells encapsulated during the scaffold fabrication process was modestly reduced with increasing PEI concentration. This study illustrates that the use of PEI during scaffold fabrication by plotting can provide an effective means to control alginate-based scaffold properties for tissue engineering applications, but that the many effects of PEI must be balanced for optimal outcomes in different situations.