Heritable, multinucleotide deletions in plants using viral delivery of a repair exonuclease and guide RNAs.

CRISPR/Cas9-mediated mutagenesis typically results in short insertion/deletion mutations, which are often too small to disrupt the function of cis-acting regulatory elements. Here, we describe a highly efficient in planta gene editing approach called VirTREX2-HLDel that achieves heritable multinucleotide deletions in both protein-coding genes and noncoding DNA regulatory elements. VirTREX2-HLDel uses RNA viruses to deliver both the 3 prime repair exonuclease 2 (TREX2) and single-guide RNAs. Our method enables recovery of multiplexed heritable deletions and increases the heritable gene editing frequency at poorly edited sites. We identified functional conservation and divergence of MICRORNA164 (miR164) in Nicotiana benthamiana and tomato (Solanum lycopersicum) using VirTREX2-HLDel and observed previously uncharacterized phenotypes in plants with large deletions at this locus. Our viral delivery method reduces the need for tissue culture and will accelerate the understanding of protein-coding and regulatory regions in plants.

Osteochondral fluid transport in an ex vivo system.

OBJECTIVE: Alterations to bone-to-cartilage fluid transport may contribute to the development of osteoarthritis (OA). Larger biological molecules in bone may transport from bone-to-cartilage (e.g., insulin, 5 kDa). However, many questions remain about fluid transport between these tissues. The objectives of this study were to (1) test for diffusion of 3 kDa molecular tracers from bone-to-cartilage and (2) assess potential differences in bone-to-cartilage fluid transport between different loading conditions. DESIGN: Osteochondral cores extracted from bovine femurs (N = 10 femurs, 10 cores/femur) were subjected to either no-load (i.e., pure diffusion), pre-load only, or cyclic compression (5 ± 2% or 10 ± 2% strain) in a two-chamber bioreactor. The bone was placed into the bone compartment followed by a 3 kDa dextran tracer, and tracer concentrations in the cartilage compartment were measured every 5 min for 120 min. Tracer concentrations were analyzed for differences in beginning, peak, and equilibrium concentrations, loading effects, and time-to-peak tracer concentration. RESULTS: Peak tracer concentration in the cartilage compartment was significantly higher compared to the beginning and equilibrium tracer concentrations. Cartilage-compartment tracer concentration and maximum fluorescent intensity were influenced by strain magnitude. No time-to-peak relationship was found between strain magnitudes and cartilage-compartment tracer concentration. CONCLUSION: This study shows that bone-to-cartilage fluid transport occurs with 3 kDa dextran molecules. These are larger molecules to move between bone and cartilage than previously reported. Further, these results demonstrate the potential impact of cyclic compression on osteochondral fluid transport. Determining the baseline osteochondral fluid transport in healthy tissues is crucial to elucidating the mechanisms OA pathology.

Heat Conduction Simulation of Chondrocyte-Embedded Agarose Gels Suggests Negligible Impact of Viscoelastic Dissipation on Temperature Change.

Agarose is commonly used for 3D cell culture and to mimic the stiffness of the pericellular matrix of articular chondrocytes. Although it is known that both temperature and mechanical stimulation affect the metabolism of chondrocytes, little is known about the thermal properties of agarose hydrogels. Thermal properties of agarose are needed to analyze potential heat production by chondrocytes induced by various experimental stimuli (carbon source, cyclical compression, etc). Utilizing ASTM C177, a custom-built thermal conductivity measuring device was constructed and used to calculate the thermal conductivity of 4.5% low gelling temperature agarose hydrogels. Additionally, Differential Scanning Calorimetry was used to calculate the specific heat capacity of the agarose hydrogels. Testing of chondrocyte-embedded agarose hydrogels commonly occurs in Phosphate-Buffered Saline (PBS), and thermal analysis requires the free convection coefficient of PBS. This was calculated using a 2D heat conduction simulation within MATLAB in tandem with experimental data collected for known boundary and initial conditions. The specific heat capacity and thermal conductivity of 4.5% agarose hydrogels was calculated to be 2.85 J/g°C and 0.121 W/mK, respectively. The free convection coefficient of PBS was calculated to be 1000.1 W/m 2 K. The values of specific heat capacity and thermal conductivity for agarose are similar to the reported values for articular cartilage, which are 3.20 J/g°C and 0.21 W/mK (Moghadam, et al. 2014). This suggests that in addition to 4.5% agarose hydrogels mimicking the physiological stiffness of the cartilage PCM, they can also mimic the thermal properties of articular cartilage for in vitro studies.

Metabolomic Profiling and Characterization of a Novel 3D Culture System for Studying Chondrocyte Mechanotransduction.

Articular chondrocytes synthesize and maintain the avascular and aneural articular cartilage. In vivo these cells are surrounded by a 3D pericellular matrix (PCM) containing predominantly collagen VI. The PCM protects chondrocytes and facilitates mechanotransduction, and PCM stiffness is critical in transmitting biomechanical signals to chondrocytes. Various culture systems with different hydrogels have been used to encapsulate chondrocytes for 3D culture, but many lack either the PCM or the in vivo stiffness of the cartilage matrix. Here, we demonstrate that primary chondrocytes cultured in alginate will form a pericellular matrix and display a phenotype similar to in vivo conditions. We found that primary human and bovine chondrocytes, when cultured in alginate beads with addition of sodium L-ascorbate for 7 days, had a pronounced PCM, retained their phenotype, and synthesized both collagens VI and II. This novel culture system enables alginate-encapsulated chondrocytes to develop a robust PCM thereby creating a model system to study mechanotransduction. We also observed distinct compression-induced changes in metabolomic profiles between the monolayer-agarose and alginate-released agarose-embedded chondrocytes indicating physiological changes in cell metabolism. Our data suggest that 3D preculture of chondrocytes in alginate before encapsulation in physiologically-stiff agarose leads to a pronounced development of pericellular matrix that is sustained in the presence of ascorbate. This novel model can be useful in studying the mechanism by which chondrocytes respond to cyclical compression and other types of loading simulating in vivo physiological conditions.