Polyakov, I. | bioRxiv Science

bioRxiv2020

Stalling chromophore maturation of the fluorescent protein Venus reveals the molecular basis of the final oxidation step

Fluorescent proteins (FPs) have revolutionised the life sciences but the mechanism of chromophore maturation is still not fully understood. Incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; the crystal structure reveals the presence of O2 located above a dehydrated enolate imidazolone (I) ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an "open" conformation, potentially allowing O2 access to the chromophore. Absorption spectroscopy supported by QM/MM simulations suggest that the first oxidation step involves formation of a hydroperoxyl intermediate in conjunction with dehydrogenation of the methylene bridge. A fully conjugated mature chromophore is formed through release of H2O2 upon irradiation of this intermediate, both in vitro and in vivo. The possibility of interrupting and photochemically restarting chromophore maturation, and the mechanistic insights opens up new approaches for engineering optically controlled fluorescent proteins.

biochemistry↗

bioRxiv2020

Evaluation of the Safety of Percutaneous Electrical Stimulation of the Phrenic and Femoral Nerves in a Chronic Porcine Model: A GLP Study

PurposeDiaphragm pacing has been proposed as a method to prevent ventilator-induced diaphragm dysfunction (VIDD) during mechanical ventilation (MV). The present study assessed the safety of lead deployment and control of diaphragm inspiratory work in synchrony with MV utilizing percutaneous electrical phrenic nerve stimulation (PEPNS) in a sedated and ventilated porcine model. MethodsThe ability to safely place PEPNS four-electrode leads near the femoral nerve using ultrasound visualization and electrical stimulation to guide lead placement using a through the needle (TTN) approach was assessed for 4 animals. The feasibility of using the PEPNS system to activate the diaphragm in synchrony with inspiration within a desired target Work of Breathing (WOB) between 0.3 and 0.7 joules/L over eight hours was tested using three of the four animals with the fourth used as a control. The ability to control WOB during inspiration was assessed for flow and pressure-controlled breaths using a flow and pressure sensor attached to the wye, the connector joining the inspiratory and expiratory limbs to the endotracheal tube. Overall health (moribundity) was assessed at baseline and throughout the study until Day 30 for the surviving animals. Gross pathology and histopathological studies were performed on the femoral nerves and diaphragm muscle tissue following termination of the animals at Day 30. ResultsThe lower bound estimate of the proportion of successful stimulation within the desired level of WOB was 95.1%, achieving the study endpoint. Triggering synchrony was statistically significant at <88ms (p<0.0001) with WOB able to be maintained between 0.3 and 0.7 joules/L. There was no evidence of tissue or nerve damage nor impact on overall animal health associated with lead placement or electrical stimulation. ConclusionThe PEPNS leads were found to be safe for their intended use and the PEPNS system met preestablished study endpoints for synchrony and stimulation efficacy.

bioengineering↗