de Vries, R. | Search bioRxiv

bioRxiv2019

Oxidative stress in alpha and beta cells as a selection criterion for biocompatible biomaterials

The clinical success of islet transplantation is limited by factors including acute ischemia, stress upon transplantation, and delayed vascularization. Islets experience high levels of oxidative stress due to delayed vascularization after transplantation and this can be further aggravated by their encapsulation and undesirable cell-biomaterial interactions. To identify biomaterials that would not further increase oxidative stress levels and that are also suitable for manufacturing a beta cell encapsulation device, we studied five clinically approved polymers for their effect on oxidative stress and islet (alpha and beta cell) function. We found that 300 poly(ethylene oxide terephthalate) 55/poly(butylene terephthalate) 45 (PEOT/PBT300) was more resistant to breakage and more elastic than other biomaterials, which is important for its immunoprotective function. In addition, PEOT/PBT300 did not induce oxidative stress or reduce viability in MIN6 beta cells, and even promoted protective endogenous antioxidant expression over 7 days. Importantly, PEOT/PBT300 is one of the biomaterials we studied that did not interfere with insulin secretion in human islets. These data indicate that PEOT/PBT300 may be a suitable biomaterial for an islet encapsulation device.

cell biology↗

bioRxiv2019

Real-time assembly of an artificial virus elucidated at the single-particle level

While the structure of a variety of viruses has been resolved at atomistic detail, their assembly pathways remain largely elusive. Key unresolved issues in assembly are the nature of the critical nucleus starting particle growth, the subsequent self-assembly reaction and the manner in which the viral genome is compacted. These issues are difficult to address in bulk approaches and are effectively only accessible by tracking the dynamics of assembly of individual particles in real time, as we show here. With a combination of single-molecule techniques we study the assembly into rod-shaped virus-like particles (VLPs) of artificial capsid polypeptides, de-novo designed previously. Using fluorescence optical tweezers we establish that oligomers that have pre-assembled in solution bind to our DNA template. If the oligomer is smaller than a pentamer, it performs one-dimensional diffusion along the DNA, but pentamers and larger oligomers are essentially immobile and nucleate VLP growth. Next, using real-time multiplexed acoustic force spectroscopy, we show that DNA is compacted in regular steps during VLP growth. These steps, of [~]30 nm of DNA contour length, fit with a DNA packaging mechanism based on helical wrapping of the DNA around the central protein core of the VLP. By revealing how real-time, single particle tracking of VLP assembly lays bare nucleation and growth principles, our work opens the doors to a new fundamental understanding of the complex assembly pathways of natural virus particles.

biophysics↗