Heterologous Expression and Maturation of an NADP-Dependent [NiFe]-Hydrogenase: A Key Enzyme in Biofuel Production

Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins

Practical Arduino: Cool Projects for Open Source Hardware

Create your own Arduino-based designs, gain in-depth knowledge of the architecture of Arduino, and learn the user-friendly Arduino language all in the context of practical projects that you can build yourself at home. Get hands-on experience using a variety of projects and recipes for everything from home automation to test equipment. Arduino has taken off as an incredibly popular building block among ubicomp (ubiquitous computing) enthusiasts, robotics hobbyists, and DIY home automation developers. Authors Jonathan Oxer and Hugh Blemings provide detailed instructions for building a wide rang

Effect of adrenaline on survival in out-of-hospital cardiac arrest: A randomised double-blind placebo-controlled trial

Background: There is little evidence from clinical trials that the use of adrenaline (epinephrine) in treating cardiac arrest improves survival, despite adrenaline being considered standard of care for many decades. The aim of our study was to determine the effect of adrenaline on patient survival to hospital discharge in out of hospital cardiac arrest. Methods: We conducted a double blind randomised placebo-controlled trial of adrenaline in out-of-hospital cardiac arrest. Identical study vials containing either adrenaline 1:1000 or placebo (sodium chloride 0.9%) were prepared. Patients were randomly allocated to receive 1 ml aliquots of the trial drug according to current advanced life support guidelines. Outcomes assessed included survival to hospital discharge (primary outcome), pre-hospital return of spontaneous circulation (ROSC) and neurological outcome (Cerebral Performance Category Score - CPC). Results: A total of 4103 cardiac arrests were screened during the study period of which 601 underwent randomisation. Documentation was available for a total of 534 patients: 262 in the placebo group and 272 in the adrenaline group. Groups were well matched for baseline characteristics including age, gender and receiving bystander CPR. ROSC occurred in 22 (8.4%) of patients receiving placebo and 64 (23.5%) who received adrenaline (OR = 3.4; 95% CI 2.0-5.6). Survival to hospital discharge occurred in 5 (1.9%) and 11 (4.0%) patients receiving placebo or adrenaline respectively (OR = 2.2; 95% CI 0.7-6.3). All but two patients (both in the adrenaline group) had a CPC score of 1-2. Conclusion: Patients receiving adrenaline during cardiac arrest had no statistically significant improvement in the primary outcome of survival to hospital discharge although there was a significantly improved likelihood of achieving ROSC