Action potential initiation site depends on neuronal excitation

The initiation site in neurons is where the excitatory and inhibitory inputs sum to generate action potentials. It is generally considered to be at a fixed location, typically at the axon hillock or initial segment, although action potentials, or impulses, could in theory arise at a site that shifts dynamically. The data reported here show that the initiation site can shift in a graded manner, by as much as 175 microm, depending on the level of neuronal excitation. Laser axotomy reveals that the Anterior Pagoda (AP) neuron of the leech is excitable within the synaptic neuropil before its axon bifurcates. Using an electrophysiological technique to measure relative delays in impulses arriving at different sites, we have found that depolarization, either by applied current or by synaptic input, can shift the site of impulse initiation in the cell proximally toward the soma and neurites receiving synaptic input. Impulse initiation in this region should enhance the efficacy of inputs synapsing there. Conversely, hyperpolarization can shift the initiation site distally. A shifting initiation site, therefore, may be a mechanism by which synaptic inputs can rapidly enhance or suppress the active response of the AP neuron to other synaptic inputs

A rational approach to improving titer in Escherichia coli‐based cell‐free protein synthesis reactions

Cell‐free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult‐to‐express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate‐based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high‐producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement

Living-donor liver transplantation at UCLA

Living-donor (LD) liver transplantation has been developed as an alternative to overcome the shortage of cadaver donor organs for pediatric recipients. We reviewed our experience with 9 LD transplants performed between August 25, 1993 and August 3, 1994. The median recipient age and weight were 13 months and 10 kilograms. Left lateral segments from parental donors, with aortic inflow via saphenous vein grafts, were used in all cases. At a median follow-up of 160 days, all donors were alive and well. Recipient and graft survival were both 89%. Rates of hepatic artery thrombosis, portal vein thrombosis, biliary complications, and acute rejection were 22%, 11%, 11%, and 67%, respectively. Excellent outcome can be achieved with LD liver transplantation in small children with minimal donor risk. This procedure has the potential to emerge as the preferred treatment for pediatric liver transplant candidates for whom it is an option