Blank, M. | Search bioRxiv

bioRxiv2023

High yield, low magnesium flexizyme reactions in a water-ice eutectic phase

Flexizymes enable the stoichiometric acylation of tRNAs with a variety of compounds, enabling the in vitro translation of peptides with both non-natural backbones and side chains. However, flexizyme reactions have several drawbacks, including single-turnover kinetics, high Mg(II) carryover inhibiting in vitro translation, and rapid product hydrolysis. Here we present flexizyme reactions utilizing an ice-eutectic phase, with high yields, 30X lower Mg(II), and long-term product stability. The eutectic flexizyme reactions increase the ease of use and flexibility of flexizyme aminoacylation, and increase the in vitro protein production.

synthetic biology↗

bioRxiv2022

The effect of carbamazepine on bone structure and strength in control and Osteogenesis Imperfecta (Col1a2 +/p.G610C) mice

The inherited brittle bone disease osteogenesis imperfecta (OI) is commonly caused by COL1A1 and COL1A2 mutations that disrupt the collagen I triple helix. This causes intracellular endoplasmic reticulum (ER) retention of the misfolded collagen and can result in a pathological ER stress response. A therapeutic approach to reduce this toxic mutant load could be to stimulate mutant collagen degradation by manipulating autophagy and/or ER-associated degradation. Since carbamazepine (CBZ) both stimulates autophagy of misfolded collagen X and improves skeletal pathology in a metaphyseal chondrodysplasia model, we tested the effect of CBZ on bone structure and strength in 3 week-old male OI Col1a2 +/p.G610C and control mice. Treatment for 3 or 6 weeks with CBZ, at the dose effective in metaphyseal chondrodysplasia, provided no therapeutic benefit to Col1a2 +/p.G610C mouse bone structure, strength, or composition, measured by micro-computed tomography, three point bending tests and Fourier-transform infrared microspectroscopy. In control mice however, CBZ treatment for 6 weeks impaired femur growth and led to lower femoral cortical and trabecular bone mass. These data, showing the negative impact of CBZ treatment on the developing mouse bones, raise important issues which must be considered in any human clinical applications of CBZ in growing individuals.

pathology↗