Spect perfusion imaging versus CT for predicting radiation injury to normal lung in lung cancer patients.

Objectives In non-small cell lung cancer (NSCLC) patients, to establish whether the fractional volumes of irradiated anatomic or perfused lung differed between those with and without deteriorating lung function or radiation associated lung injury (RALI).Methods 48 patients undergoing radical radiotherapy for NSCLC had a radiotherapy-planning CT scan and single photon emission CT lung perfusion imaging (99mTc-labelled macroaggregate albumin). CT defined the anatomic and the single photon emission CT scan (co-registered with CT) identified the perfused (threshold 20 % of maximum) lung volumes. Fractional volumes of anatomic and perfused lung receiving more than 5, 10, 13, 20, 30, 40, 50 Gy were compared between patients with deteriorating (>median decline) vs stable (vs stable FEV1 ( p = 0.005, 0.005 and 0.025 respectively) but did not differ for higher doses of radiation (>30, 40, 50 Gy). Fractional volumes of anatomic and perfused lung receiving > 10 Gy best predicted decline in FEV1 (Area under receiver operating characteristic curve (Az = 0.77 and 0.76 respectively); sensitivity/specificity 75%/81 and 80%/71%) for a 32.7% anatomic and 33.5% perfused volume cut-off. Irradiating an anatomic fractional volume of 4.7% to > 50 Gy had a sensitivity/specificity of 83%/89 % for indicating RALI (Az = 0.83).Conclusion A 10-20 Gy radiation dose to anatomic or perfused lung results in decline in FEV1. A fractional anatomic volume of >5% receiving >50 Gy influences development of RALI.Advances in knowledge Extent of low-dose radiation to normal lung influences functional respiratory decline

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

High level heterologous expression in E. coli using the anaerobically-activated nirB promoter.

The anaerobically-regulated nirB promoter was used to express heterologous genes in Escherichia coli. Under anaerobic conditions the promoter was able to express tetanus toxin fragment C at approximately 20% total cell protein (tcp) and the Bordetella pertussis antigen pertactin at greater than 30% tcp. These levels are comparable to those obtained for the same products using the tac promoter. The nirB promoter is very well regulated, giving almost two orders of magnitude increase in fragment C on complete removal of oxygen. The use of this anaerobically-induced promoter in the production of recombinant proteins in E. coli is discussed